Recombinant, active caspases and uses thereof

ABSTRACT

Rev-caspases comprising a primary product in which the small subunit is N-terminal to the large subunit are provided. Rev-caspases are used for screening and identifying caspase inhibitors and enhancers. Rev-caspase genes can be delivered to cells for gene therapy.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. application Ser. No. 09/227,721, filed Jan. 8, 1999; now U.S. Pat. No. 6,379,950 which application claims the benefit of priority from U.S. Provisional Application Serial No. 60/070,987, filed Jan. 9, 1998, now abandoned; all prior applications are hereby incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to regulating apoptosis, and more particularly to the novel aspartate-specific cysteine proteases known as caspases, their coding regions, mutant forms thereof, and their use in screening assays and as pharmaceutical compositions for the controlled death of targeted cells to treat human disease.

BACKGROUND OF THE INVENTION

Tissue homeostasis is maintained by the process of apoptosis—that is, the normal physiological process of programmed cell death. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.

Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes that occurs with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can alter the natural progression of many of these diseases.

Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself, is a cascade of proteolytic events analogous to that of the blood coagulation cascade.

Several gene families and products that modulate the apoptotic process have now been identified. One family is the aspartate-specific cysteine proteases (“caspases”). The caspase Ced-3, identified in C. elegans, is required for programmed cell death during development of the roundworm C. elegans. Ced-3 homologues as well as other caspases have been characterized. The human caspase family includes, for example, human ICE (interleukin-1-β converting enzyme) (caspase-1), ICE_(rel)II (caspase-4), ICE_(rel)III (caspase-5), Mch5 (caspase-8), Mch4 (caspase-10), ICE-LAP6 (caspase-9), Mch2 (caspase-6), CPP32 (caspase-3), ICE-LAP3 (casepase-7), ICH-1 (caspase-2), Caspase 11-14, and others.

The caspases share many features. In this regard, caspases are cysteine proteases (named for a cysteine residue in the active site) that cleave substrates at Asp-X bonds. Furthermore, the primary caspase product is a zymogen that requires proteolytic cleavage at specific internal aspartate residues for activation. The primary gene product is arranged such that the N-terminal peptide (prodomain) precedes a large subunit domain, which precedes a small subunit domain. Cleavage of a caspase yields two subunits, a large (generally approximately 20 kD) and a small (generally approximately 10 kD) subunit that associate non-covalently: to form a heterodimer, and, in some caspases, an N-terminal peptide of varying length (see FIG. 1). The heterodimer may combine non-covalently to form a tetramer.

Caspase zymogens are themselves substrates for caspases. Inspection of the interdomain linkages in each zymogen reveals target sites (i.e. protease sites) that indicate a hierarchical relationship of caspase activation. By analyzing such pathways, it has been demonstrated that caspases are required for apoptosis to occur. Moreover, caspases appear to be necessary for the accurate and limited proteolytic events which are the hallmark of classic apoptosis (see Salvesen and Dixit, Cell, 91: 443-446, 1997). However, when overexpressed in mammalian cells, the short prodomain caspases-3 and -6 cells are unable to undergo autocatalytic processing/activation and do not induce apoptosis. Thus, no cellular model system has been developed in which to test inhibitors of these caspases nor is gene delivery of a caspase commonplace.

Therefore, there exists a need in the art for methods of assaying compounds for their ability to affect caspase activity as well as for methods of regulating caspases in order to treat diseases and syndromes. The present invention provides recombinant caspase constructs that are active in cells, allowing the regulation of apoptosis for the treatment of pathology as well as providing methods and compositions for assaying compounds for caspase inhibitory and, thus, anti-apoptotic effects, while further providing other related advantages.

SUMMARY OF THE INVENTION

The present invention generally provides rev-caspases. In one aspect, the invention provides an isolated nucleic acid molecule encoding a rev-caspase. In certain embodiments, the rev-caspase is selected from the group consisting of rev-caspase-1, rev-caspase-2, rev-caspase-3, rev-caspase-4, rev-caspase-5, rev-caspase-6, rev-caspase-7, rev-caspase-8, rev-caspase-9, rev-caspase-10, rev-caspase-11, rev-caspase-12, rev-caspase-13, and rev-caspase-14. In other preferred embodiments, the rev-caspase is a human rev-caspase. Nucleic acid and amino acid sequences of rev-caspases are provided. The invention also provides rev-caspase proteins.

In another aspect, an expression vector comprising the nucleic acid molecule encoding rev-caspase is provided, wherein the sequence encoding rev-caspase is operatively linked to a promoter. In certain embodiments, the promoter is inducible, such as HIV LTR. Host cells transfected with the expression vectors are also provided.

In the present invention, methods of identifying an inhibitor or enhancer of caspase processing activity are provided, comprising: (a) contacting a sample containing an in vitro translated rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

In other aspects, methods are provided for identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a cell transfected with the vector expressing rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

Methods are also provided for identifying an inhibitor or enhancer of caspase-mediated apoptosis, comprising: (a) contacting a cell transfected with the vector expressing rev-caspase with a candidate inhibitor or candidate enhancer or with a reference compound; and (b) detecting cell viability, wherein viability of cells contacted with a candidate is increased in the presence of an inhibitor and is decreased in the presence of an enhancer compared to cells contacted with a reference compound.

In other aspects, gene delivery vehicles, comprising the nucleic acid molecule encoding a rev-caspase are provided, wherein the rev-caspase sequence is operatively linked to a promoter. In preferred embodiments, the gene delivery vehicle is a retrovirus or adenovirus or the nucleic acid molecule is associated with a polycation. The gene delivery vehicle may further comprise a ligand that binds a cell surface receptor.

The invention also provides methods of treating cancer or autoimmune diseases, comprising administering to a patient the gene delivery vehicles disclosed herein.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, the various references set forth below that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the processing and folding of human caspase-3 into the mature zymogen represented by bar and ribbon diagrams and is representative of other caspases. α helices are shown as spirals and the β strands are represented by arrows. The N- and the C-termini of the LS are labeled N-LS and C-LS, respectively, and the termini of the SS are similarly labeled N-SS and C-SS.

FIGS. 1B and C are schematic representations of rev-caspase-3 and -6, respectively. In both FIG. 1B and C the N-terminus of the SS and the C-terminus of the LS are labeled as in FIG. 1A and the linker region between the C-SS and the N-LS which includes the caspase-3 or -6 prodomain is represented by a thin line. Solid arrows indicate the cleavage sites (DEVDG (SEQ ID NO:38), Asp9 and Asp28—for rev-caspase-3 and VEIDA (SEQ ID NO:116), and Asp23—for rev-caspase-6) within the linker region. The hatched boxes represent a 15 residue-long T7-tag on the N-termini of the wild-type and the rev-caspases. All aspartate processing sites are indicated on the bar diagrams. FIG. 1B further depicts a schematic representation of the spontaneous folding of rev-caspase-3 into the mature zymogen represented a ribbon diagram. The ribbon diagram of rev-caspase-3 is based on the published crystal structure of caspase-3.

FIG. 2A is a scanned image of an autoradiogram representing SDS-PAGE analysis of rev-caspase autoprocessing. Caspase-3 and -6 or their rev-versions (Rev) including active site Cys to Ala mutants (Rev C/A) in pRSC-lacZ constructs were in vitro translated in the presence of ³⁵S-methionine. The translation products were then analyzed by SDS-PAGE and autoradiography. The LS and the SS are indicated.

FIGS. 2B and C are scanned images of autoradiograms representing SDS-PAGE analysis of the autoprocessing of rev-caspase-3 and -6, respectively in the presence of varying levels of selected caspase inhibitors. Rev-caspase-3 (FIG. 2B) or rev-caspase-6 (FIG. 2C) were in vitro translated in the presence of increasing concentrations of DEVD-CHO (0.04 μM); (SEQ ID NO:52), or zVAD-fmk (0-5 μM). The translation products were then analyzed as in FIG. 2A. WT, wild-type.

FIGS. 3A and B are scanned images representing the SDS-PAGE analysis of the ability of rev-caspase-3 (imaged by western blot) and -6 (imaged by autoradiogram) to cleave PARP and lamin, respectively. In FIG. 3A purified human PARP was incubated with buffer (lane 1) or BL-21 bacterial extracts prepared from bacteria transformed with caspase-3 (lane 2), rev-caspase-3 (lane 3), caspase-6 (lane 4), rev-caspase-6 (lane 5) constructs or empty pET28a vector (lane 6) for 2 h at 37° C. The reaction products were then analyzed by SDS-PAGE and Western blotting with anti-human PARP antibody. In FIG. 3B a cDNA encoding the C-terminus of lamin A (amino acids) which contain the caspase-6 cleavage site (VEIDA); (SEQ ID NO:116), was amplified by PCR and in vitro translated in the presence of ³⁵S-methionine. The labeled product was incubated with buffer (lane 1) or the BL-21 bacterial extracts listed above for 2 h at 37° C., and then analyzed by SDS-PAGE and autoradiography. The cleavage products are indicated to the right.

FIGS. 4A and B are bar diagrams representing the ability of rev-caspase-3 and -6 to induce apoptosis in MCF-7 cells. MCF-7 cells were transiently transfected with either rev-caspase-3 (FIG. 4A), or rev-caspase-6 (FIG. 4B) expression constructs in combination with 4-fold of CrmA, p35 or Bcl-2 expression constructs, or 20 μM zVAD-fmk. Cells transfected with an empty vector or the wild-type caspase-3 or -6 were used as controls.

FIG. 5A is a scanned image of an autoradiogram representing SDS-PAGE analysis of the enzymatic activity of uncleavable rev-caspase-3. Uncleavable rev-caspase-3 was in vitro translated in the absence or the presence of increasing concentrations of DEVD-CHO (SEQ ID NO:52),. The translation product contains a cleavable 35 residues-long His6-T7-tag at its N-terminus. The active site mutant rev-caspase-3 (Rev C/A) was used as a control. The p32 cleavage product without the His6-T7-tag is indicated to the right.

FIG. 5B is a plot of an activity assay of bacterially expressed uncleavable rev-caspase-3. The plot measures the ability of the uncleavable rev-caspase-3 to cleave the DEVD-AMC (SEQ ID NO:52), substrate. Rev, rev-caspase-3; Rev-mod, uncleavable rev-caspase-3; Rev-C/A, rev-caspase-3 with an active site mutation.

FIG. 6 is a multiple amino acid sequence alignment of the relatively conserved regions of the caspases (SEQ ID NOs:54-115). In the bottom line, “c” refers to residues involved in catalysis, “b” refers to residues that bind the substrate-carboxylate of P1 Asp, “a” refers to residues adjacent to the substrate P2-P4 recognition responsible amino acids, “DX” indicates known and potential processing sites between the small and large subunits of the caspases. The roman numerals at the left of the figure indicate the caspase subfamilies: Ced-like (I), ICE-like (II), and the Nedd2/Ich-1-like (III). The asterisk represents the non-conservative substitution in the active site pentapeptide sequences of Mch4 (caspase-10), Mch5 (caspase-8), and Mch6 (caspase-9)

FIG. 7 depicts a nucleotide sequence of Rev-caspase-3 (SEQ ID NO:1).

FIG. 8 depicts a nucleotide sequence of uncleavable Rev-caspase-3 (SEQ ID NO:2).

FIG. 9 depicts a nucleotide sequence of Rev-caspase-6 (SEQ ID NO:3).

FIG. 10 depicts a schematic of some possible rev-caspases. 1, intervening sequence; SS, small subunit; P, prodomain; LS, large subunit; X, linker.

FIGS. 11A and 11B depict a nucleotide (SEQ ID NOs:4 and 5) and predicted amino acid sequence of caspase-1 (SEQ ID NO:6).

FIGS. 12A and 12B depict a nucleotide (SEQ ID NOs:7 and 8) and predicted amino acid sequence of caspase-2 (SEQ ID NO:9).

FIGS. 13A and 13B depict a nucleotide (SEQ ID NOs:10 and 11) and predicted amino acid sequence of caspase-3 (SEQ ID NO:12).

FIGS. 14A and 14B depict a nucleotide (SEQ ID NOs:13 and 14) and predicted amino acid sequence of caspase-4 (SEQ ID NO:15).

FIGS. 15A and 15B depict a nucleotide (SEQ ID NOs:16 and 17) and predicted amino acid sequence of caspase-5 (SEQ ID NO:18).

FIGS. 16A and 16B depict a nucleotide (SEQ ID NOs:19 and 20) and predicted amino acid sequence of caspase-6 (SEQ ID NO:21).

FIGS. 17A and 17B depict a nucleotide (SEQ ID NOs:22 and 23) and predicted amino acid sequence of caspase-7 (SEQ ID NO:24).

FIGS. 18A-18C depict a nucleotide (SEQ ID NOs:25 and 26) and predicted amino acid sequence of caspase-8 (SEQ ID NO:27).

FIGS. 19A and 19B depict a nucleotide (SEQ ID NOs:28 and 29) and predicted amino acid sequence of caspase-9 (SEQ ID NO:30).

FIGS. 20A and 20B depict a nucleotide (SEQ ID NOs:31 and 32) and predicted amino acid sequence of caspase-10 (SEQ ID NO:33).

FIGS. 21A, 21B, and 21C depict predicted amino acid sequences of Rev-caspase-3 (A; SEQ ID NO:34), uncleavable rev-caspase-3 (B; SEQ ID NO:35), and rev-caspase-6 (C; SEQ ID NO:36).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.

As used herein, a caspase refers to a cysteine protease that specifically cleaves proteins after Asp residues. Caspases are initially expressed as zymogens, in which a large subunit is N-terminal to a small subunit. Caspases are generally activated by cleavage at internal Asp residues (FIG. 1A). These proteins have been identified in many eukaryotes, including C. elegans, Drosophila, mouse, and humans. Currently, there are at least 14 known caspase genes, named caspase-1 through caspase-14. Caspases are found in myriad organisms, including human, mouse, insect (e.g., Drosophila), and other invertebrates (e.g., C. elegans). In Table 1, ten human caspases are listed along with their alternative names. The nucleotide and amino acid sequences of representative human caspase gene products are presented in SEQ ID NOs:4-33 and FIGS. 11-20.

Caspase Alternative name Caspase-1 ICE Caspase-2 ICH-1 Caspase-3 CPP32, Yama, apopain Caspase-4 ICE_(rel)II; TX, ICH-2 Caspase-5 ICE_(rel)III; TY Caspase-6 Mch2 Caspase-7 Mch3, ICE-LAP3, CMH-1 Caspase-8 FLICE; MACH; Mch5 Caspase-9 ICE-LAP6; Mch6 Caspase-10 Mch4, FLICE-2

As used herein, “rev-caspase” refers to a cysteine protease that specifically cleaves proteins after Asp residues and is expressed as a zymogen, in which a small subunit is N-terminal to a large subunit.

Within the context of this invention, it should be understood that a caspase or rev-caspase includes wild-type protein sequences, as well as other variants (including alleles) of the native protein sequence. Briefly, such variants may result from natural polymorphisms or may be synthesized by recombinant methodology, and differ from wild-type protein by one or more amino acid substitutions, insertions, deletions, or the like. Typically, when engineered, amino acid substitutions will be conservative, i.e., substitution of amino acids within groups of polar, non-polar, aromatic, charged, etc. amino acids. In the region of homology to the native sequence, variants should preferably have at least 90% amino acid sequence identity, and within certain embodiments, greater than 92%, 95%, or 97% identity.

As will be appreciated by those skilled in the art, a nucleotide sequence encoding a caspase, rev-caspase or variant may differ from the known native sequences, due to codon degeneracies, nucleotide polymorphisms, or amino acid differences. In other embodiments, variants should preferably hybridize to the native nucleotide sequence at conditions of normal stringency, which is approximately 25-30° C. below Tm of the native duplex (e.g., 5×SSPE, 0.5% SDS, 5×Denhardt's solution, 50% formamide, at 42° C. or equivalent conditions; see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Low stringency hybridizations utilize conditions approximately 40° C. below Tm, and high stringency hybridizations utilize conditions approximately 10° C. below Tm. Variants preferably have at least 75% nucleotide identity to native sequence, preferably at least 80%, 85%, and most preferably at least 90% nucleotide identity.

An “isolated nucleic acid molecule” refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been separated from its source cell (including the chromosome it normally resides in) at least once in a substantially pure form. Nucleic acid molecules may be comprised of a wide variety of nucleotides, including DNA, RNA, nucleotide analogues, or some combination of these.

A. Caspase and Rev-caspase Genes and Gene Products

As noted above, the invention provides compositions relating to caspase and rev-caspase genes and gene products, and methods for the use of the genes and gene products. In particular, the invention provides rev-caspase constructs that are active when expressed in cells. Given the disclosure provided herein, a caspase gene can be isolated from a variety of cell types and engineered to produce a rev-caspase.

1. Isolation of Caspase Genes

The present invention, as described herein, provides rev-caspase genes, which are constructed from caspase genes. Caspase genes may be isolated from either genomic DNA or preferably cDNA. Isolation of caspase genes from genomic DNA or cDNA typically can proceed by, first, generating an appropriate DNA library through techniques for constructing libraries that are known in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989) or purchased from commercial sources (e.g., Clontech, Palo Alto, Calif.). Briefly, cDNA libraries can be constructed in bacteriophage vectors (e.g., λZAPII), plasmids, or others, which are suitable for screening, while genomic DNA libraries can be constructed in chromosomal vectors, such as YACs (yeast artificial chromosomes), bacteriophage vectors, such as λEMBL3, λgt10, cosmids, or plasmids.

In one embodiment known caspase sequences may be utilized to design an oligonucleotide hybridization probe suitable for screening genomic or cDNA libraries. Preferably, such oligonucleotide probes are 20-30 bases in length. To facilitate hybridization detection, the oligonucleotide may be conveniently labeled, generally at the 5′ end, with a reporter molecule, such as a radionuclide, (e.g., ³²P), enzymatic label, protein label, fluorescent label, or biotin. Such libraries are then generally plated as phage or colonies, depending upon the vector used. Subsequently, a nitrocellulose or nylon membrane, to which the colonies or phage have been transferred, is probed to identify candidate clones which contain the caspase gene. Such candidates may be verified as containing caspase DNA by any of various means including, for example, DNA sequence analysis or hybridization with a second, non-overlapping probe.

Once a library is identified as containing a caspase gene, the gene can be isolated by amplification. Briefly, when using cDNA library DNA as a template amplification primers are designed based upon known caspase gene sequences (see GenBank Accession Nos. X65019 (caspase-1), U13021 (caspase-2), U13737 (caspase-3), U25804 (caspase-4), U28015 (caspase-5), U20536 (caspase-6), U37448 (caspase-7), U60520 (caspase-8), U56390 (caspase-9), U60519 (caspase-10) Y13089 (caspase-11), Y13090 (caspase-12), AF078533 (caspase-13), AF092997 (caspase-14), and sequences provided herein). Amplification of cDNA libraries made from cells with high caspase activity is preferred. Primers for amplification are preferably derived from sequences in the 5′ and 3′ untranslated region in order to isolate a full-length cDNA. The primers preferably have a GC content of about 50% and contain restriction sites to facilitate cloning and do not have self-complementary sequences nor do they contain complementary sequences at their 3′ end (to prevent primer-dimer formation). The primers are annealed to cDNA or genomic DNA and sufficient amplification cycles are performed to yield a product readily visualized by gel electrophoresis and staining. The amplified fragment is purified and inserted into a vector, such as λgt10 or pBS(M13+), and propagated. Confirmation of the nature of the fragment is obtained by DNA sequence analysis or indirectly through amino acid sequencing of the encoded protein.

Other methods may also be used to obtain a caspase encoding nucleic acid molecule. For example, a nucleic acid molecule encoding caspase may be obtained from an expression library by screening with an antibody or antibodies reactive to caspase (see, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1987; Ausubel, et al. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, NY, 1995).

Caspase genes from a variety of species may be isolated using the compositions provided herein. For closely related species, the human sequence or portion thereof may be utilized as a probe on a genomic or cDNA library. For example, a fragment of caspase that encompasses the catalytic site may be labeled and used as a probe on a library constructed from mouse, primate, rat, dog, or other vertebrate, warm-blooded or mammalian species. An initial hybridization at normal stringency may yield candidate clones or fragments. If no hybridization is initially observed, varying degrees of stringency may be used. (see Sambrook et al. supra, and other well-known sources for stringency conditions) While such probes may also be used to probe libraries from evolutionarily diverse species, such as Drosophila, hybridization conditions will likely be more relaxed.

While relaxed hybridization conditions using probes designed from human sequences may identify caspase genes of evolutionarily diverse species it may be more beneficial to attempt to directly isolate these genes from a library using methods which do not require the human sequence per se. These methods include, but are not limited to, amplification using primers derived from conserved areas, amplification using degenerate primers from various regions, antibody probing of expression libraries, and the like. For example, random-primed amplification (e.g., polymerase chain reaction) may be employed (see, e.g., Methods Enzymol. 254: 275, 1995; Trends Genet. 11: 242, 1995; Liang and Pardee, Science 257: 967, 1992; Welsh et al., Nucl. Acids Res. 20: 4965, 1992). In addition, variations of random-primed PCR may also be used, especially when a particular gene or gene family is desired. In such a method, one of the amplification primers is an “anchored oligo(dT) (oligo(dT)dN)” and the other primer is a degenerate primer based upon amino acid or nucleotide sequence of a related gene. A gene sequence is identified as a caspase by amino acid similarity and/or nucleic acid similarity. Generally, amino acid similarity is preferred. Candidate caspase genes are examined for enzyme activity by one of the functional assays described herein or other equivalent assays.

Variants of caspase and rev-caspase genes provided herein may be engineered from natural variants (e.g., polymorphisms, splice variants, mutants), synthesized or constructed. Many methods have been developed for generating mutants (see, generally, Sambrook et al., supra; Ausubel, et al., supra, and the discussion above). Briefly, preferred methods for generating a few nucleotide substitutions utilize an oligonucleotide that spans the base or bases to be mutated and contains the mutated base or bases. The oligonucleotide is hybridized to complementary single stranded nucleic acid and second strand synthesis is primed from the oligonucleotide. The double-stranded nucleic acid is prepared for transformation into host cells, typically E. coli, but alternatively, other prokaryotes, yeast or other eukaryotes. Standard screening and vector growth protocols are used to identify mutant sequences and obtain high yields.

Similarly, deletions and/or insertions of the caspase or rev-caspase genes may be constructed by any of a variety of known methods as discussed supra. For example, the gene can be digested with restriction enzymes and religated such that a sequence is deleted or religated with additional sequences such that an insertion or large substitution is made. Other means of generating variant sequences may be employed with methods known in the art, for example those described in Sambrook et al. (supra) and Ausubel et al. (supra). Verification of variant sequences is typically accomplished by restriction enzyme mapping, sequence analysis, or probe hybridization. Variants which catalyze Asp-specific cleavages are useful in the context of this invention.

B. Rev-Caspases

The caspases of the present invention are generated by rearranging the gene sequence of the caspase gene such that the nucleic acid sequence encoding the small subunit precedes (is 5′ to) the nucleic acid sequence encoding the large subunit. These rearranged caspases are called rev-caspases.

1. Structure of Rev-caspases

The rev-caspases of the present invention comprise at least a portion of the small subunit and at least a portion of the large subunit. In preferred embodiments, the prodomain or a portion thereof (see FIGS. 1, 10) and/or an intervening sequence or a portion thereof (see FIG. 10) are also present in rev-caspase. In other preferred embodiments, a “linker” region is located between the small and large subunits.

The boundaries of the small subunit and large subunit are identified either experimentally by amino acid sequence analysis of the mature caspase or by inspection of structural homology (e.g., the conserved Asp-X cleavage site). For exemplary purposes, the Table below presents the boundaries of the prodomain (P), large subunit (LS), intervening sequence (I), and small subunit (SS) of human caspase-1 through -10. The nucleotide numbers refer to the nucleotides in SEQ ID NOs:4-33 and in FIGS. 11-20.

Intervening Caspase Prodomain Large Subunit sequence Small Subunit Caspase-1 1-357 358-891  892-948 949-1212 Caspase-2 1-456 457-948  949-990 991-1305 Caspase-3 1-84  85-525 526-831  Caspase-4 1-240 241-810  811-867 868-1131 Caspase-5 1-363 364-933  934-1254 Caspase-6 1-69  70-537 538-579 580-879  Caspase-7 1-69  70-594 595-909  Caspase-8 1-681 682-1173 1174-1488  Caspase-9 1-390 391-945  946-990 991-1248 Caspase-10 1-657 658-1116 1117-1437 

As noted above, a portion of the large subunit and small subunit may be used in rev-caspase constructs. When designing rev-caspases that contain a portion of these subunits, the active site (e.g., QACXG, where X is Arg, Gln, or Gly); (SEQ ID NO:51), which is located near the C-terminus of the large subunit should not be deleted if protease activity is desired. Preferably, the 3-dimensional structure as determined by X-ray crystallography (see Mittl et al., J. Biol. Chem., 272: 6539-6547, 1997; Rotondu et al., Nat. Struct. Biol., 3: 619-625, 1996; Walker et al., Cell, 78: 343-352, 1994; Wilson et al., Nature, 370: 270-275, 1994) is maintained. For example, from the x-ray crystallographic structures of caspases, the amino acids, that are important in binding substrates have been identified. Likewise, substitutions of amino acids in the active site may be detrimental to maintaining activity. Although it is preferred that both subunits are derived from the same caspase, combinations of subunits from different caspases and/or from different species may be used.

The prodomain (sometimes called an N-terminal peptide) is generally not required for enzyme activity and is normally released in vivo. Rev-caspases of the present invention optionally have a prodomain or portion thereof. Similarly, the intervening sequence, which is present in certain caspases, is optional for inclusion in rev-caspases.

In certain embodiments, a linker region is engineered between the small and large subunits. A “linker region”, as used herein, refers to a peptide of from about 5 to about 50 amino acids. In preferred embodiments, the linker may contain a protease sensitive or cleavage site. Any site recognized by an intracellular protease may be used. In addition, multiple protease sensitive sites may be tandemly arranged in the same linker. Preferred protease sensitive sites are susceptible to cleavage by caspases or by viral proteases. Preferred caspase sensitive sites include, but are not limited to DXXDG (wherein X is any amino acid; SEQ ID NO:37); DEVDG (SEQ ID NO:38), IETDG (SEQ ID NO:39), YVADG (SEQ ID NO:40), YVHDG (SEQ ID NO:41), and WEHDG (SEQ ID NO:42). Furthermore, the Gly residue may be Ala or another small amino acid. The latter three sites are specifically cleaved by caspases-1, -4, and -5. Other sites specifically cleaved by only one or a few caspases are preferred in certain embodiments. Viral proteases cleavage sites include, but are not limited to, those recognized and cleaved by HIV protease, HCV (hepatitis C virus) protease, HBV (hepatitis B virus) protease, and rhinovirus protease.

2. Construction of Rev-caspases

Rev-caspases may be constructed from caspase sequences by a variety of methods known in the art. A preferred method is amplification (e.g., polymerase chain reaction (PCR)) to selectively amplify the individual subunits and place these in cloning vectors such as pUC such as described in Example 1. Moreover, such PCR reactions can be performed in a variety of ways such that the primers used for amplification contain specific restriction endonuclease sites to facilitate insertion into a vector.

Further, a variety of other methodologies besides PCR may be used to attain the desired rearrangement. For example, one skilled in the art may employ isothermal methods to amplify the nucleotide sequence of interest, using existing restriction endonuclease sites present in the nucleotide sequence to excise and insert sequences, or by the introduction of distinct restriction endonuclease sites by site-directed mutagenesis followed by excision and insertion. These and other methods are described in Sambrook et al., supra; Ausubel, et al., supra. Briefly, one methodology is to generate single-stranded cDNA of the caspase of interest, followed by annealing a primer, which is complementary except for the desired alteration (e.g., a small insertion, deletion, or mutation such that a unique restriction site is created between the large and small subunits and/or at the 5′ and 3′ ends of both subunits). Bacterial cells are transformed and screened for those which contain desired construct. This construct is then digested to liberate the subunit sequences, which can then be purified and religated into the appropriate orientation.

As indicated above, rev-caspase genes may be manipulated to contain insertions, deletions or substitutions. Moreover, such variant rev-caspase genes useful in the context of this invention include those which facilitate Asp-specific cleavages indicative of caspase activity. Further, variants which are incapable of being cleaved into separate subunits are encompassed within the context of this invention, if those variants are able to facilitate Asp-specific cleavages by way of a cysteine-containing active site. By way of guidance, amino acids involved in catalysis, Asp recognition in substrate, and P2-P4 substrate recognition are provided in FIG. 6.

C. Vectors, Host Cells and Means of Expressing and Producing Protein

Caspase may be expressed in a variety of host organisms. In certain embodiments, caspase is produced in bacteria, such as E. coli, or mammalian cells (e.g., CHO and COS-7), for which many expression vectors have been developed and are available. Other suitable host organisms include other bacterial species, and eukaryotes, such as yeast (e.g., Saccharomyces cerevisiae), and insect cells (e.g., Sf9).

A DNA sequence encoding rev-caspase is introduced into an expression vector appropriate for the host. In certain embodiments, rev-caspase is inserted into a vector such that a fusion protein is produced. The rev-caspase sequence is derived as described herein. As discussed above, the sequence may contain alternative codons for each amino acid with multiple codons. The alternative codons can be chosen as “optimal” for the host species. Restriction sites are typically incorporated into the primer sequences and are chosen with regard to the cloning site of the vector. If necessary, translational initiation and termination codons can be engineered into the primer sequences.

At minimum, the vector must contain a promoter sequence. As used herein, a “promoter” refers to a nucleotide sequence that contains elements that direct the transcription of a linked gene. At minimum, a promoter contains an RNA polymerase binding site. More typically, in eukaryotes, promoter sequences contain binding sites for other transcriptional factors that control the rate and timing of gene expression. Such sites include TATA box, CAAT box, POU box, AP1 binding site, and the like. Promoter regions may also contain enhancer elements. When a promoter is linked to a gene so as to enable transcription of the gene, it is “operatively linked”.

Other regulatory sequences may be included. Such sequences include a transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences are operationally associated with one another to allow transcription or translation.

The expression vectors used herein include a promoter designed for expression of the proteins in a host cell (e.g., bacterial). Suitable promoters are widely available and are well known in the art. Inducible or constitutive promoters are preferred. Such promoters for expression in bacteria include promoters from the T7 phage and other phages, such as T3, T5, and SP6, and the trp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used. Promoters for expression in eukaryotic cells include the P10 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784), MMTV LTR, CMV IE promoter, RSV LTR, SV40, metallothionein promoter (see, e.g., U.S. Pat. No. 4,870,009) and the like.

The promoter controlling transcription of rev-caspase may itself be controlled by a repressor. In some systems, the promoter can be derepressed by altering the physiological conditions of the cell, for example, by the addition of a molecule that competitively binds the repressor, or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli lacI repressor responsive to IPTG induction, the temperature sensitive λc1857 repressor, and the like. The E. coli lacI repressor is preferred.

In other preferred embodiments, the vector also includes a transcription terminator sequence. A “transcription terminator region” has either a sequence that provides a signal that terminates transcription by the polymerase that recognizes the selected promoter and/or a signal sequence for polyadenylation.

Preferably, the vector is capable of replication in the host cells. Thus, when the host cell is a bacterium, the vector preferably contains a bacterial origin of replication. Preferred bacterial origins of replication include the fl-ori and col E1 origins of replication, especially the ori derived from pUC plasmids. In yeast, ARS or CEN sequences can be used to assure replication. A well-used system in mammalian cells is SV40 ori.

The plasmids also preferably include at least one selectable marker that is functional in the host. A selectable marker gene includes any gene that confers a phenotype on the host that allows transformed cells to be identified and selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp^(r)), tetracycline resistance gene (Tc^(r)) and the kanamycin resistance gene (Kan^(r)). The kanamycin resistance gene is presently preferred. Suitable markers for eukaryotes usually require a complementary deficiency in the host (e.g., thymidine kinase (tk) in tk-hosts). However, drug markers are also available (e.g., G418 resistance and hygromycin resistance).

The sequence of nucleotides encoding rev-caspase may also include a secretion signal, whereby the resulting peptide is a precursor protein processed and secreted. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium. Secretion signals suitable for use are widely available and are well known in the art (von Heijne, J. Mol. Biol. 184: 99-105, 1985). Prokaryotic and eukaryotic secretion signals that are functional in E. coli (or other host) may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: pelB (Lei et al., J. Bacteriol. 169: 4379, 1987), phoA, ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase.

One skilled in the art appreciates that there are a wide variety of suitable vectors for expression in bacterial cells and which are readily obtainable. Vectors such as the pET series (Novagen, Madison, Wis.), the tac and trc series (Pharmacia, Uppsala, Sweden), pTTQ18 (Amersham International plc, England), pACYC 177, pGEX series, and the like are suitable for expression of a rev-caspase. Baculovirus vectors, such as pBlueBac (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may be used for expression in insect cells, such as Spodoptera frugiperda sf9 cells (see, U.S. Pat. No. 4,745,051). The choice of a bacterial host for the expression of a rev-caspase is dictated in part by the vector. Commercially available vectors are paired with suitable hosts.

A wide variety of suitable vectors for expression in eukaryotic cells are available. Such vectors include pCMVLacI, pXT1 (Stratagene Cloning Systems, La Jolla, Calif.); pCDNA series, pREP series, pEBVHis (Invitrogen, Carlsbad, Calif.). In certain embodiments, rev-caspase gene is cloned into a gene targeting vector, such as pMClneo, a pOG series vector (Stratagene Cloning Systems).

Rev-caspase is isolated by standard methods, such as affinity chromatography, size exclusion chromatography, metal ion chromatography, ionic exchange chromatography, HPLC, and other known protein isolation methods. (see generally Ausubel et al. supra; Sambrook et al. supra). An isolated purified protein gives a single band on SDS-PAGE when stained with Coomassie blue.

Rev-caspase may be expressed as a hexa-his fusion protein and isolated by metal-containing chromatography, such as nickel-coupled beads. Briefly, a sequence encoding His₆ is linked to a DNA sequence encoding a rev-caspase. Although the His₆ sequence can be positioned anywhere in the molecule, preferably it is linked at the 3′ end immediately preceding the termination codon. The fusion may be constructed by any of a variety of methods. A convenient method is amplification of the rev-caspase gene using a downstream primer that contains the codons for His₆.

Purified rev-caspase protein may be used in assays to screen for inhibitory drugs. These assays may be performed in vitro or in vivo and utilize any of the methods described herein or that are known in the art. The protein may also be crystallized and subjected to X-ray analysis to determine its 3-dimensional structure or used to raise antibodies.

D. Uses of Rev-caspase Gene and Gene Product

1. Inhibitors and Enhancers of Caspase Activity

Candidate inhibitors and enhancers may be isolated or procured from a variety of sources, such as bacteria, fungi, plants, parasites, libraries of chemicals, peptides or peptide derivatives and the like. Inhibitors and enhancers may be also be rationally designed, based on the protein structure determined from X-ray crystallography (see, Mittl et al., J. Biol. Chem., 272: 6539-6547, 1997). In certain preferred embodiments, the inhibitor targets a specific caspase (e.g., caspase-3 and not any other caspases).

Without being held to a particular mechanism, the inhibitor may act by preventing processing of caspase or by preventing enzymatic activity, or by other mechanism. The inhibitor may act directly or indirectly. In preferred embodiments, inhibitors interfere in the processing of the caspase protein. In other preferred embodiments, the inhibitors are small molecules. In a most preferred embodiment, the inhibitors prevent apoptosis. Inhibitors should have a minimum of side effects and are preferably non-toxic. Inhibitors that can penetrate cells are preferred.

In addition, enhancers of caspase activity or expression are desirable in certain circumstances. At times, increasing apoptosis will have a therapeutic effect. For example, tumors or cells that mediate autoimmune diseases are appropriate cells for destruction. Enhancers may increase the rate or efficiency of caspase processing, increase transcription or translation, or act through other mechanisms. As is apparent to one skilled in the art, many of the guidelines presented above apply to the design of enhancers as well.

Screening assays for inhibitors and enhancers will vary according to the type of inhibitor or enhancer and the nature of the activity that is being affected. Assays may be performed in vitro or in vivo. In general, in vitro assays are designed to evaluate caspase protein processing or caspase enzymatic activity, and in vivo assays are designed to evaluate caspase protein processing, caspase enzymatic activity, apoptosis, or caspase cleavage of substrate. In any of the assays, a statistically significant increase or decrease compared to a proper control is indicative of enhancement or inhibition.

One in vitro assay can be performed by examining the effect of a candidate compound on processing of rev-caspase into two subunits. Briefly, a cleavable form of rev-caspase, that is a primary translation product, is obtained from an in vitro translation system. The cleavable form of rev-caspase is preferably constructed to be auto-cleaved, but can be constructed to be cleaved by other protease components present or added to the reaction. This primary product is contacted with or without or translated in the presence or absence of a candidate compound and assessed for appearance of the two subunits to facilitate detection, typically, the primary product of rev-caspase is labeled during translation, cell viability, and the like. The two subunits may be readily detected by autoradiography after gel electrophoresis. One skilled in the art will recognize that other methods of labeling and detection may be used alternatively.

An alternative in vitro assay is designed to measure cleavage of a caspase substrate (e.g., Acetyl DEVD-aminomethyl coumarin (amc) (SEQ ID NO:52), lamin, PRPP, and the like). Substrate turnover may be assayed using either cleavable or noncleavable rev-caspase. Briefly, in this method, rev-caspase is translated and allowed sufficient time to be processed, if a cleavable rev-caspase is being used. The caspase substrate along with the candidate compound is added to the reaction. Detection of cleaved substrate is performed by any one of a variety of standard methods. Generally, the substrate will be labeled and followed by an appropriate detection means.

Moreover, any known enzymatic analysis can be used to follow the inhibitory or enhancing ability of a candidate compound with regard to a rev-caspase of this invention. For example, one could express the rev-caspase of interest in a cell line be it bacterial, insect, mammalian or other, either in cleavable or noncleavable form and purify the rev-caspase. The purified rev-caspase could then be used in a variety of assays to follow its catalytic ability in the presence of candidate compounds, as noted above. Such methods of expressing and purifying recombinant proteins are known in the art and examples can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989 as well as in a number of other sources.

In vivo assays are typically performed in cells transfected either transiently or stably with an expression vector containing a rev-caspase gene, such as those described herein. These cells are used to measure rev-caspase processing, substrate turnover, or apoptosis in the presence or absence of a candidate compound. When assaying apoptosis, a variety of cell analyses may be used including, for example, dye staining and microscopy to examine nucleic acid fragmentation and porosity of the cells. Further, in vivo assaying for the ability of the transfected rev-caspase to cleave known substrates that are co-transfected or placed in the cell culture media in the presence of the candidate compound can be performed thereby allowing for the detection and determination of substrate turnover.

The assays briefly described herein may be used to identify an enhance or inhibitor that is specific for an individual caspase. In a preferred embodiment candidate compounds would be analyzed using a variety of rev-caspases (e.g., rev-caspase-1 through rev-caspase-14) to identify specific for individual caspases.

A variety of methodologies exist can be used to investigate the effect of a candidate compound. Such methodologies are those commonly used to analyze enzymatic reactions and include, for example, SDS-PAGE, spectroscopy, HPLC analysis, autoradiography, chemiluminescence, chromogenic reactions, and immunochemistry (e.g., blotting, precipitating, etc.).

Inhibitors and enhancers may be used in the context of this invention to exert control over the cell death process or cytokine activation (e.g., IL-1, which is activated by caspase-1). Thus, these inhibitors and enhancers will have utility in diseases characterized by either excessive or insufficient levels of apoptosis. Inhibitors of proteases have potential to treat the major neurodegenerative diseases: stroke, Parkinson's Disease, Alzheimer's Disease, and ALS. As well, caspase protease inhibitors may be used to inhibit apoptosis in the heart following myocardial infarction, in the kidney following acute ischemia, and in diseases of the liver. In other embodiments, inhibitors of caspase-1 can be used to inhibit the release of the pro-inflammatory IL-1β, and thus may provide therapeutic benefit in treating inflammation and/or autoimmune disorders. Enhancers of caspase activity may be used in contexts when apoptosis or cytokine activation are desired. For example, inducing or increasing apoptosis in cancer cells or aberrantly proliferating cells may be effected by delivery of a caspase enhancer.

The inhibitors and enhancers may be further coupled with a targeting moiety that binds a cell surface receptor specific to the cells. Administration of inhibitors or enhancers will generally follow established protocols. The compounds of the present invention may be administered either alone, or as a pharmaceutical composition. Briefly, pharmaceutical compositions of the present invention may comprise one or more of the inhibitors or enhancers as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and preservatives. In addition, pharmaceutical compositions of the present invention may also contain one or more additional active ingredients.

Compositions of the present invention may be formulated for the manner of administration indicated, including for example, for oral, nasal, venous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. Within other embodiments of the invention, the compositions described herein may be administered as part of a sustained release implant. Within yet other embodiments, compositions of the present invention may be formulized as a lyophilizate, utilizing appropriate excipients which provide stability as a lyophilizate, and subsequent to rehydration.

2. Gene Therapy

As noted above, rev-caspases may be delivered to cells as part of gene delivery vehicles. In many diseases and syndromes, too little apoptosis is an important feature in their development. Treatment of many autoimmune diseases and tumors would benefit from increased apoptosis. One means to increase apoptosis is to provide target cells with caspase genes in an expressible form. This may be accomplished by delivery of DNA or cDNA capable of in vivo transcription of the rev-caspase. More specifically, in order to produce rev-caspases in vivo, a nucleic acid sequence coding for the rev-caspase is placed under the control of a eukaryotic promoter (e.g., a pol III promoter, CMV or SV40 promoter). Where it is desired to more specifically control transcription, the rev-caspase may be placed under the control of a tissue or cell specific promoter (e.g., to target cells in the liver), or an inducible promoter, such as metallothionein.

Many techniques for introduction of nucleic acids into cells are known. Such methods include retroviral vectors and subsequent retrovirus infection, adenoviral or adeno-associated viral vectors and subsequent infection, and complexes of nucleic acid with a condensing agent (e.g., poly-lysine). These complexes or viral vectors may be targeted to particular cell types by way of a ligand incorporated into the vehicle. Many ligands specific for tumor cells and other cells are well known in the art.

A wide variety of vectors may be utilized within the context of the present invention, including for example, plasmids, viruses, retrotransposons and cosmids. Representative examples include adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Yei et al., Gene Therapy 1: 192-200, 1994; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J Neurosci. 5(10):1287-1291, 1993), adeno-associated type 1 (“AAV-1”) or adeno-associated type 2 (“AAV-2”) vectors (see WO 95/13365; Flotte et al., PNAS 90(22):10613-10617, 1993), hepatitis delta vectors, live, attenuated delta viruses and herpes viral vectors (e.g., U.S. Pat. No. 5,288,641), as well as vectors which are disclosed within U.S. Pat. No. 5,166,320. Other representative vectors include retroviral vectors (e.g., EP 0 415 731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218.

Within certain aspects of the invention, nucleic acid molecules that encode the rev-caspase may be introduced into a host cell utilizing a vehicle, or by various physical methods. Representative examples of such methods include transformation using calcium phosphate precipitation (Dubensky et al., PNAS 81: 7529-7533, 1984), direct microinjection of such nucleic acid molecules into intact target cells (Acsadi et al., Nature 352: 815-818, 1991), and electroporation whereby cells suspended in a conducting solution are subjected to an intense electric field in order to transiently polarize the membrane, allowing entry of the nucleic acid molecules. Other procedures include the use of nucleic acid molecules linked to an inactive adenovirus (Cotton et al., PNAS 89: 6094, 1990), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1989), microprojectile bombardment (Williams et al., PNAS 88: 2726-2730, 1991), polyeation compounds such as polylysine, receptor specific ligands, liposomes entrapping the nucleic acid molecules, spheroplast fusion whereby E. coli containing the nucleic acid molecules are stripped of their outer cell walls and fused to animal cells using polyethylene glycol, viral transduction, (Cline et al., Pharmac. Ther. 29: 69, 1985; and Friedmann et al., Science 244: 1275, 1989), and DNA ligand (Wu et al, J. of Biol. Chem. 264: 16985-16987, 1989), as well as psoralen inactivated viruses such as Sendai or Adenovirus. In one embodiment, the rev-caspase construct is introduced into the host cell using a liposome.

As noted above, pharmaceutical compositions also are provided by this invention. These compositions may contain any of the above described inhibitors, enhancers, DNA molecules, vectors or host cells, along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.

In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example intraarticularly, intracranially, intradermally, intrahepatically, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor. In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition. Pharmaceutical compositions are useful for both diagnostic or therapeutic purposes.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. Dosages may be determined most accurately during clinical trials. Patients may be monitored for therapeutic effectiveness by appropriate technology, including signs of clinical exacerbation, imaging and the like.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES Example 1 Generation of cDNAs Expressing Rev-caspase-3 and 6 Precursors

Generation of cDNAs encoding rev-caspase-3 and 6 precursors were generated by PCR. The large (LS) and small subunits (SS) of caspase-3 were amplified with the following primers using the caspase-3 cDNA as a template:

LS-forward, ATGGAGAACACTGAAAACTCAG (SEQ ID NO:43); LS-reverse, GTCATCATCAACACCTCAGTCT (SEQ ID NO:44); SS-forward, GGATCCATGATTGAGACAGACAGTGG (SEQ ID NO:45); SS-reverse, ATCAACTTCATCGTGATAAAAATAGAGTTC (SEQ ID NO:46).

The PCR products were cloned separately into the Sma I site of pBluescript KS⁺. The small subunit was then excised from KS⁺-vector with Bam HI and inserted into the Bam HI site of the second KS⁺-vector which contains the large subunit. This places the small subunit in-frame 5′ to the large subunit. Rev-caspase-6 was amplified and cloned in the KS⁺-vector in a similar way. The following PCR primers were used with caspase-6-His6 cDNA as a template:

LS-forward, ATGAGCTCGGCCTCGGGG (SEQ ID NO:47); LS-reverse, TTAATCTACTACATCCAAAGG (SEQ ID NO:48); SS-forward, GGATCCATGGTAGAAATAGATGCAGCCTCCGTTTAC (SEQ ID NO:49) SS-reverse, ATCAATTTCAACGTGGTGGTGGTGGTGGTGC (SEQ ID NO:50).

The resulting nucleotide sequences were such that the wild type subunit order was reversed thus creating a contiguous nucleotide sequence wherein the coding region for the small subunit preceded that of the large subunit (See FIG. 1). The engineered contiguous caspase-3 and 6 molecules (i.e., rev-caspase molecules) in which the SS was fused in frame N-terminal to the LS, and a cleavage site (DEVDG in the case of caspase-3; VEIDS in the case of caspase-6 (these internal cleavage sites were designed to be specific for the caspase in which it was introduced in order to investigate the autocatalytic activity of the particular caspase)) was introduced between the two subunits and is depicted in FIG. 1B and C.

To express the rev-caspases in bacteria, their cDNAs were excised with Bam HI/Xho I and subcloned into the bacterial expression vector pET28a (Novagen, Inc.) in-frame with the T7-tag of this vector.

Example 2 Expression of Rev-caspase in Mammlian Cells and Assay for Apoptosis

To express the rev-caspases in mammalian cells and assay their apoptotic activity, they were amplified with the T7-tag primer and the LS-reverse primers using the pET28a constructs as templates, and subcloned into the mammalian double expression vector pRSC-LacZ (MacFarlane et al., J. Biol. Chem., 272: 25417-25420, 1997; Tsang et al., Bio/Technology, 22: 68, 1997). This vector allows the expression of lacZ under the Rous Sarcoma virus promoter, and the test cDNA under the CMV promoter. To assay for apoptosis, MCF-7 or 293 cells were transfected, using the method commercially available as the Lipofect Amine method (Life Technologies, Inc.), with the pRSC-LacZ constructs in the presence or absence of different apoptosis-inhibitors. 30 h after transfection cells were stained with β-galactosidase and examined for morphological signs of apoptosis. The percentage of round blue apoptotic cells (mean±SD) were represented as a function of total blue cells under each condition (n≧3).

Example 3 In Vitro Translation of Caspases

³⁵S-labeled caspases (wild-type and rev-caspases) were obtained by in vitro translation in the presence of ³⁵S-methionine using a coupled transcription/translation system in rabbit reticulocyte lysate using TNT Kit (Promega) according to the manufacturer's recommendations. Unlike the wild-type caspase-3 and 6, FIG. 2A demonstrates that rev-caspase-3 and 6 were able to undergo autocatalytic processing in the in vitro translation reaction. Further, this processing was completely inhibited by mutation of the active site Cys of rev-caspase-3 and 6 (FIG. 2A, lanes 3 and 6) and by selected caspase inhibitors (See Example 4). Because the in vitro translated products are present at very low concentration in the reaction mixture, the observed cleavage must be attributed to an intramolecular processing within the caspase heterotetramer.

Example 4 Effects of Inhibitors on Rev-caspase-3 and 6 Activity

To test the effect of selected caspase inhibitors on the autocatalytic activity of rev-caspase-3 and 6, the rev-caspases were translated as in Example 3, but in the presence of varying amounts of inhibitors. As demonstrated by FIG. 2B, in the presence of increasing amounts of DEVD-CHO (SEQ ID NO:52), a decrease in the amount of cleavage products and a corresponding increase in the amount of the revcaspase-3 precursor was observed. This corresponded to nearly 50-90% inhibition of the autocatalytic activity of rev-caspase-3 at 40-400 nM concentration. However, the same concentrations of this inhibitor had little effect on the autocatalytic activity of rev-caspase-6 (FIG. 2C). This is consistent with earlier observations that caspase-6 is poorly inhibited by DEVD-CHO (SEQ ID NO:52); see Srinivasula et al., J. Biol. Chem., 271: 27099-27106, 1996. On the other hand, as is apparent from inspection of FIGS. 2B and 2C, z-VAD-fmk had nearly an equal inhibitory effect on rev-caspase-3 and 6 autocatalytic activity at the concentration used in this experiment. Nevertheless, nearly 10-fold more of z-VAD-fink than DEVD-CHO (SEQ ID NO:52) was required to obtain complete inhibition of caspase-3 activity. Similarly, baculovirus p35 had nearly an equal inhibitory effect on rev-caspase-3 and 6 autocatalytic activity (data not shown).

Example 5 Caspase-3 and 6 Specificity Retention of Bacterially Expressed Rev-caspase-3 and -6

At limited caspase concentrations, poly(ADP) ribose polymerase (PARP) is specifically cleaved by caspase-3 and 7 but not other caspases. Similarly, lamin is specifically cleaved by caspase-6 but not other caspases. To compare the activity of the wild-type and rev-caspase-3 and 6 towards PARP and lamin, rev-caspase-3 and 6 were expressed in bacteria and then incubated with the two substrates PARP and lamin. As shown in FIGS. 3A and B, the activity of the rev-caspases towards these two substrates were indistinguishable from their wild-type counterparts. Both caspase-3 variants (rev and WT), but not caspase-6 variants efficiently cleaved PARP. In contrast, both caspase-6 variants, but not caspase-3 variants efficiently cleaved lamin. These results demonstrate that the mature caspases generated from the rev and the wild type constructs have identical substrate specificity.

Example 6 Induction of Apoptosis in Mammalian Cells by Rev-caspase-3 and -6

To determine the apoptotic activity of rev-caspase-3 and 6 in vivo, the rev-caspases were expressed in human MCF-7 cells, transfected as explained above in Example 2. As evidenced by FIGS. 4A and B, unlike the wild type caspase-3 and 6, the rev-caspases potently induced apoptosis in nearly 90% of the transfected cells. Overexpression of Bc1-2 or CrmA, which protect against different forms of apoptosis, did not significantly reduce their apoptotic activity. Nevertheless, overexpression of the baculovirus p35, which inhibits the activity of most caspases, partially protected against their apoptotic activity. Also, incubation of the transfected cells in the presence of 100 μM z-VAD-fmk, dramatically reduced their apoptotic activity to nearly 30%. These data demonstrate directly that the activity of caspase-3 and 6 are downstream of the CrmA and Bcl-2 block in the apoptotic cascade, and can only be inhibited by high concentration of the pancaspase-inhibitor z-VAD-fmk.

Example 7 Activity of Noncleavable Rev-caspase-3

To demonstrate that the rev-caspase molecules are inherently active and do not require separation of the two subunits and that the two subunits are derived from the same contiguous molecule, the DEVD (SEQ ID NO:52) site was removed and Asp9 and 28, that are present between the two subunits of rev-caspase-3, were mutated (see FIG. 2A). However, to follow the activity of this molecule a cleavable 35 residue long His6-T7-tag N-terminal to the IETD (SEQ ID NO:53) site was introduced (see FIG. 1B). FIG. 5 demonstrates that upon in vitro translation of this molecule, as described in Example 3 above, there was no evidence of cleavage between the two subunits. Nevertheless, the translated molecule was active as evident from its ability to cleave its T7-tag to form the p32 species (FIG. 5). In the presence of 400 nM DEVD-CHO (SEQ ID NO:52), processing of the T7-tag was inhibited and only the full length p34 species can be seen. Furthermore, expression of this molecule into MCF-7 cells potently induced apoptosis in these cells. These data demonstrate that when the two subunits of a caspase are rearranged in the reverse order, it is not necessary to separate them from each other to generate an active caspase. Thus by mimicking the mature caspase structure, it is possible to design a contiguous active caspase molecules.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

                   #             SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 116 <210> SEQ ID NO 1 <211> LENGTH: 873 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Rev-Caspase-3  contructed  #from human caspase -3 <400> SEQUENCE: 1 atgattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat ac #cagtggag     60 gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aa #attcaaag    120 gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cg #acaagctt    180 gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tg #agtccttt    240 tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc ca #tgctcaca    300 aaagaactct atttttatca cgatgaagtt gatgggggat cccccatgga ga #acactgaa    360 aactcagtgg attcaaaatc cattaaaaat ttggaaccaa agatcataca tg #gaagcgaa    420 tcaatggact ctggaatatc cctggacaac agttataaaa tggattatcc tg #agatgggt    480 ttatgtataa taattaataa taagaatttt cataagagca ctggaatgac at #ctcggtct    540 ggtacagatg tcgatgcagc aaacctcagg gaaacattca gaaacttgaa at #atgaagtc    600 aggaataaaa atgatcttac acgtgaagaa attgtggaat tgatgcgtga tg #tttctaaa    660 gaagatcaca gcaaaaggag cagttttgtt tgtgtgcttc tgagccatgg tg #aagaagga    720 ataatttttg gaacaaatgg acctgttgac ctgaaaaaaa taacaaactt tt #tcagaggg    780 gatcgttgta gaagtctaac tggaaaaccc aaacttttca ttattcaggc ct #gccgtggt    840 acagaactgg actgtggcat tgagacagac tga        #                   #        873 <210> SEQ ID NO 2 <211> LENGTH: 858 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Uncleavalbe Rev-Caspase-3   #contructed from       human caspase -3 <400> SEQUENCE: 2 atgattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat ac #cagtggag     60 gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aa #attcaaag    120 gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cg #acaagctt    180 gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tg #agtccttt    240 tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc ca #tgctcaca    300 aaagaactct atttttatca cggatccccc atggagaaca ctgaaaactc ag #tggcttca    360 aaatccatta aaaatttgga accaaagatc atacatggaa gcgaatcaat gg #cctctgga    420 atatccctgg acaacagtta taaaatggat tatcctgaga tgggtttatg ta #taataatt    480 aataataaga attttcataa gagcactgga atgacatctc ggtctggtac ag #atgtcgat    540 gcagcaaacc tcagggaaac attcagaaac ttgaaatatg aagtcaggaa ta #aaaatgat    600 cttacacgtg aagaaattgt ggaattgatg cgtgatgttt ctaaagaaga tc #acagcaaa    660 aggagcagtt ttgtttgtgt gcttctgagc catggtgaag aaggaataat tt #ttggaaca    720 aatggacctg ttgacctgaa aaaaataaca aactttttca gaggggatcg tt #gtagaagt    780 ctaactggaa aacccaaact tttcattatt caggcctgcc gtggtacaga ac #tggactgt    840 ggcattgaga cagactga              #                   #                   # 858 <210> SEQ ID NO 3 <211> LENGTH: 903 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Rev-Caspase-6 contructed fro #m human caspase -3 <400> SEQUENCE: 3 atggtagaaa tagatgcagc ctccgtttac acgctgcctg ctggagctga ct #tcctcatg     60 tgttactctg ttgcagaagg atattattct caccgggaaa ctgtgaacgg ct #catggtac    120 attcaagatt tgtgtgagat gttgggaaaa tatggctcct ccttagagtt ca #cagaactc    180 ctcacactgg tgaacaggaa agtttctcag cgccgagtgg acttttgcaa ag #acccaagt    240 gcaattggaa agaagcaggt tccctgtttt gcctcaatgc taactaaaaa gc #tgcatttc    300 tttccaaaat ctaatctcga gcaccaccac caccaccacg ttgaaattga tg #ggggatcc    360 cccatgagct cggcctcggg gctccgcagg gggcacccgg caggtgggga ag #aaaacatg    420 acagaaacag atgccttcta taaaagagaa atgtttgatc cggcagaaaa gt #acaaaatg    480 gaccacagga ggagaggaat tgctttaatc ttcaatcatg agaggttctt tt #ggcactta    540 acactgccag aaaggcgggg cacctgcgca gatagagaca atcttacccg ca #ggttttca    600 gatctaggat ttgaagtgaa atgctttaat gatcttaaag cagaagaact ac #tgctcaaa    660 attcatgagg tgtcaactgt tagccacgca gatgccgatt gctttgtgtg tg #tcttcctg    720 agccatggcg aaggcaatca catttatgca tatgatgcta aaatcgaaat tc #agacatta    780 actggcttgt tcaaaggaga caagtgtcac agcctggttg gaaaacccaa ga #tatttatc    840 atccaggcat gtcggggaaa ccagcacgat gtgccagtca ttcctttgga tg #tagtagat    900 taa                   #                   #                   #            903 <210> SEQ ID NO 4 <211> LENGTH: 1215 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 4 atggccgaca aggtcctgaa ggagaagaga aagctgttta tccgttccat gg #gtgaaggt     60 acaataaatg gcttactgga tgaattatta cagacaaggg tgctgaacaa gg #aagagatg    120 gagaaagtaa aacgtgaaaa tgctacagtt atggataaga cccgagcttt ga #ttgactcc    180 gttattccga aaggggcaca ggcatgccaa atttgcatca catacatttg tg #aagaagac    240 agttacctgg cagggacgct gggactctca gcagatcaaa catctggaaa tt #accttaat    300 atgcaagact ctcaaggagt actttcttcc tttccagctc ctcaggcagt gc #aggacaac    360 ccagctatgc ccacatcctc aggctcagaa gggaatgtca agctttgctc cc #tagaagaa    420 gctcaaagga tatggaaaca aaagtcggca gagatttatc caataatgga ca #agtcaagc    480 cgcacacgtc ttgctctcat tatctgcaat gaagaatttg acagtattcc ta #gaagaact    540 ggagctgagg ttgacatcac aggcatgaca atgctgctac aaaatctggg gt #acagcgta    600 gatgtgaaaa aaaatctcac tgcttcggac atgactacag agctggaggc at #ttgcacac    660 cgcccagagc acaagacctc tgacagcacg ttcctggtgt tcatgtctca tg #gtattcgg    720 gaaggcattt gtgggaagaa acactctgag caagtcccag atatactaca ac #tcaatgca    780 atctttaaca tgttgaatac caagaactgc ccaagtttga aggacaaacc ga #aggtgatc    840 atcatccagg cctgccgtgg tgacagccct ggtgtggtgt ggtttaaaga tt #cagtagga    900 gtttctggaa acctatcttt accaactaca gaagagtttg aggatgatgc ta #ttaagaaa    960 gcccacatag agaaggattt tatcgctttc tgctcttcca caccagataa tg #tttcttgg   1020 agacatccca caatgggctc tgtttttatt ggaagactca ttgaacatat gc #aagaatat   1080 gcctgttcct gtgatgtgga ggaaattttc cgcaaggttc gattttcatt tg #agcagcca   1140 gatggtagag cgcagatgcc caccactgaa agagtgactt tgacaagatg tt #tctacctc   1200 ttcccaggac attaa               #                   #                   #  1215 <210> SEQ ID NO 5 <211> LENGTH: 1215 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 5 taccggctgt tccaggactt cctcttctct ttcgacaaat aggcaaggta cc #cacttcca     60 tgttatttac cgaatgacct acttaataat gtctgttccc acgacttgtt cc #ttctctac    120 ctctttcatt ttgcactttt acgatgtcaa tacctattct gggctcgaaa ct #aactgagg    180 caataaggct ttccccgtgt ccgtacggtt taaacgtagt gtatgtaaac ac #ttcttctg    240 tcaatggacc gtccctgcga ccctgagagt cgtctagttt gtagaccttt aa #tggaatta    300 tacgttctga gagttcctca tgaaagaagg aaaggtcgag gagtccgtca cg #tcctgttg    360 ggtcgatacg ggtgtaggag tccgagtctt cccttacagt tcgaaacgag gg #atcttctt    420 cgagtttcct atacctttgt tttcagccgt ctctaaatag gttattacct gt #tcagttcg    480 gcgtgtgcag aacgagagta atagacgtta cttcttaaac tgtcataagg at #cttcttga    540 cctcgactcc aactgtagtg tccgtactgt tacgacgatg ttttagaccc ca #tgtcgcat    600 ctacactttt ttttagagtg acgaagcctg tactgatgtc tcgacctccg ta #aacgtgtg    660 gcgggtctcg tgttctggag actgtcgtgc aaggaccaca agtacagagt ac #cataagcc    720 cttccgtaaa cacccttctt tgtgagactc gttcagggtc tatatgatgt tg #agttacgt    780 tagaaattgt acaacttatg gttcttgacg ggttcaaact tcctgtttgg ct #tccactag    840 tagtaggtcc ggacggcacc actgtcggga ccacaccaca ccaaatttct aa #gtcatcct    900 caaagacctt tggatagaaa tggttgatgt cttctcaaac tcctactacg at #aattcttt    960 cgggtgtatc tcttcctaaa atagcgaaag acgagaaggt gtggtctatt ac #aaagaacc   1020 tctgtagggt gttacccgag acaaaaataa ccttctgagt aacttgtata cg #ttcttata   1080 cggacaagga cactacacct cctttaaaag gcgttccaag ctaaaagtaa ac #tcgtcggt   1140 ctaccatctc gcgtctacgg gtggtgactt tctcactgaa actgttctac aa #agatggag   1200 aagggtcctg taatt               #                   #                   #  1215 <210> SEQ ID NO 6 <211> LENGTH: 404 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 6 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Ly #s Leu Phe Ile Arg Ser  1               5   #                10   #                15 Met Gly Glu Gly Thr Ile Asn Gly Leu Leu As #p Glu Leu Leu Gln Thr             20       #            25       #            30 Arg Val Leu Asn Lys Glu Glu Met Glu Lys Va #l Lys Arg Glu Asn Ala         35           #        40           #        45 Thr Val Met Asp Lys Thr Arg Ala Leu Ile As #p Ser Val Ile Pro Lys     50               #    55               #    60 Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Ty #r Ile Cys Glu Glu Asp 65                   #70                   #75                   #80 Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Al #a Asp Gln Thr Ser Gly                 85   #                90   #                95 Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Va #l Leu Ser Ser Phe Pro             100       #           105       #           110 Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Me #t Pro Thr Ser Ser Gly         115           #       120           #       125 Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Gl #u Glu Ala Gln Arg Ile     130               #   135               #   140 Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Il #e Met Asp Lys Ser Ser 145                 1 #50                 1 #55                 1 #60 Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Gl #u Glu Phe Asp Ser Ile                 165   #               170   #               175 Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Th #r Gly Met Thr Met Leu             180       #           185       #           190 Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Ly #s Lys Asn Leu Thr Ala         195           #       200           #       205 Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Al #a His Arg Pro Glu His     210               #   215               #   220 Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Me #t Ser His Gly Ile Arg 225                 2 #30                 2 #35                 2 #40 Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gl #n Val Pro Asp Ile Leu                 245   #               250   #               255 Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Th #r Lys Asn Cys Pro Ser             260       #           265       #           270 Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gl #n Ala Cys Arg Gly Asp         275           #       280           #       285 Ser Pro Gly Val Val Trp Phe Lys Asp Ser Va #l Gly Val Ser Gly Asn     290               #   295               #   300 Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu As #p Asp Ala Ile Lys Lys 305                 3 #10                 3 #15                 3 #20 Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cy #s Ser Ser Thr Pro Asp                 325   #               330   #               335 Asn Val Ser Trp Arg His Pro Thr Met Gly Se #r Val Phe Ile Gly Arg             340       #           345       #           350 Leu Ile Glu His Met Gln Glu Tyr Ala Cys Se #r Cys Asp Val Glu Glu         355           #       360           #       365 Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gl #n Pro Asp Gly Arg Ala     370               #   375               #   380 Gln Met Pro Thr Thr Glu Arg Val Thr Leu Th #r Arg Cys Phe Tyr Leu 385                 3 #90                 3 #95                 4 #00 Phe Pro Gly His <210> SEQ ID NO 7 <211> LENGTH: 1308 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 7 atggccgctg acaggggacg caggatattg ggagtgtgtg gcatgcatcc tc #atcatcag     60 gaaactctaa aaaagaaccg agtggtgcta gccaaacagc tgttgttgag cg #aattgtta    120 gaacatcttc tggagaagga catcatcacc ttggaaatga gggagctcat cc #aggccaaa    180 gtgggcagtt tcagccagaa tgtggaactc ctcaacttgc tgcctaagag gg #gtccccaa    240 gcttttgatg ccttctgtga agcactgagg gagaccaagc aaggccacct gg #aggatatg    300 ttgctcacca ccctttctgg gcttcagcat gtactcccac cgttgagctg tg #actacgac    360 ttgagtctcc cttttccggt gtgtgagtcc tgtccccttt acaagaagct cc #gcctgtcg    420 acagatactg tggaacactc cctagacaat aaagatggtc ctgtctgcct tc #aggtgaag    480 ccttgcactc ctgaatttta tcaaacacac ttccagctgg catataggtt gc #agtctcgg    540 cctcgtggcc tagcactggt gttgagcaat gtgcacttca ctggagagaa ag #aactggaa    600 tttcgctctg gaggggatgt ggaccacagt actctagtca ccctcttcaa gc #ttttgggc    660 tatgacgtcc atgttctatg tgaccagact gcacaggaaa tgcaagagaa ac #tgcagaat    720 tttgcacagt tacctgcaca ccgagtcacg gactcctgca tcgtggcact cc #tctcgcat    780 ggtgtggagg gcgccatcta tggtgtggat gggaaactgc tccagctcca ag #aggttttt    840 cagctctttg acaacgccaa ctgcccaagc ctacagaaca aaccaaaaat gt #tcttcatc    900 caggcctgcc gtggagatga gactgatcgt ggggttgacc aacaagatgg aa #agaaccac    960 gcaggatccc ctgggtgcga ggagagtgat gccggtaaag aaaagttgcc ga #agatgaga   1020 ctgcccacgc gctcagacat gatatgcggc tatgcctgcc tcaaagggac tg #ccgccatg   1080 cggaacacca aacgaggttc ctggtacatc gaggctcttg ctcaagtgtt tt #ctgagcgg   1140 gcttgtgata tgcacgtggc cgacatgctg gttaaggtga acgcacttat ca #aggatcgg   1200 gaaggttatg ctcctggcac agaattccac cggtgcaagg aaatgtctga at #actgcagc   1260 actctgtgcc gccacctcta cctgttccca ggacaccctc ccacatga   #              1308 <210> SEQ ID NO 8 <211> LENGTH: 1308 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 8 taccggcgac tgtcccctgc gtcctataac cctcacacac cgtacgtagg ag #tagtagtc     60 ctttgagatt ttttcttggc tcaccacgat cggtttgtcg acaacaactc gc #ttaacaat    120 cttgtagaag acctcttcct gtagtagtgg aacctttact ccctcgagta gg #tccggttt    180 cacccgtcaa agtcggtctt acaccttgag gagttgaacg acggattctc cc #caggggtt    240 cgaaaactac ggaagacact tcgtgactcc ctctggttcg ttccggtgga cc #tcctatac    300 aacgagtggt gggaaagacc cgaagtcgta catgagggtg gcaactcgac ac #tgatgctg    360 aactcagagg gaaaaggcca cacactcagg acaggggaaa tgttcttcga gg #cggacagc    420 tgtctatgac accttgtgag ggatctgtta tttctaccag gacagacgga ag #tccacttc    480 ggaacgtgag gacttaaaat agtttgtgtg aaggtcgacc gtatatccaa cg #tcagagcc    540 ggagcaccgg atcgtgacca caactcgtta cacgtgaagt gacctctctt tc #ttgacctt    600 aaagcgagac ctcccctaca cctggtgtca tgagatcagt gggagaagtt cg #aaaacccg    660 atactgcagg tacaagatac actggtctga cgtgtccttt acgttctctt tg #acgtctta    720 aaacgtgtca atggacgtgt ggctcagtgc ctgaggacgt agcaccgtga gg #agagcgta    780 ccacacctcc cgcggtagat accacaccta ccctttgacg aggtcgaggt tc #tccaaaaa    840 gtcgagaaac tgttgcggtt gacgggttcg gatgtcttgt ttggttttta ca #agaagtag    900 gtccggacgg cacctctact ctgactagca ccccaactgg ttgttctacc tt #tcttggtg    960 cgtcctaggg gacccacgct cctctcacta cggccatttc ttttcaacgg ct #tctactct   1020 gacgggtgcg cgagtctgta ctatacgccg atacggacgg agtttccctg ac #ggcggtac   1080 gccttgtggt ttgctccaag gaccatgtag ctccgagaac gagttcacaa aa #gactcgcc   1140 cgaacactat acgtgcaccg gctgtacgac caattccact tgcgtgaata gt #tcctagcc   1200 cttccaatac gaggaccgtg tcttaaggtg gccacgttcc tttacagact ta #tgacgtcg   1260 tgagacacgg cggtggagat ggacaagggt cctgtgggag ggtgtact   #              1308 <210> SEQ ID NO 9 <211> LENGTH: 435 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 9 Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gl #y Val Cys Gly Met His  1               5   #                10   #                15 Pro His His Gln Glu Thr Leu Lys Lys Asn Ar #g Val Val Leu Ala Lys             20       #            25       #            30 Gln Leu Leu Leu Ser Glu Leu Leu Glu His Le #u Leu Glu Lys Asp Ile         35           #        40           #        45 Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Al #a Lys Val Gly Ser Phe     50               #    55               #    60 Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pr #o Lys Arg Gly Pro Gln 65                   #70                   #75                   #80 Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Gl #u Thr Lys Gln Gly His                 85   #                90   #                95 Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gl #y Leu Gln His Val Leu             100       #           105       #           110 Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Le #u Pro Phe Pro Val Cys         115           #       120           #       125 Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Le #u Ser Thr Asp Thr Val     130               #   135               #   140 Glu His Ser Leu Asp Asn Lys Asp Gly Pro Va #l Cys Leu Gln Val Lys 145                 1 #50                 1 #55                 1 #60 Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Ph #e Gln Leu Ala Tyr Arg                 165   #               170   #               175 Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Va #l Leu Ser Asn Val His             180       #           185       #           190 Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Se #r Gly Gly Asp Val Asp         195           #       200           #       205 His Ser Thr Leu Val Thr Leu Phe Lys Leu Le #u Gly Tyr Asp Val His     210               #   215               #   220 Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gl #n Glu Lys Leu Gln Asn 225                 2 #30                 2 #35                 2 #40 Phe Ala Gln Leu Pro Ala His Arg Val Thr As #p Ser Cys Ile Val Ala                 245   #               250   #               255 Leu Leu Ser His Gly Val Glu Gly Ala Ile Ty #r Gly Val Asp Gly Lys             260       #           265       #           270 Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Ph #e Asp Asn Ala Asn Cys         275           #       280           #       285 Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Ph #e Ile Gln Ala Cys Arg     290               #   295               #   300 Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gl #n Asp Gly Lys Asn His 305                 3 #10                 3 #15                 3 #20 Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Al #a Gly Lys Glu Lys Leu                 325   #               330   #               335 Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Me #t Ile Cys Gly Tyr Ala             340       #           345       #           350 Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Th #r Lys Arg Gly Ser Trp         355           #       360           #       365 Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Gl #u Arg Ala Cys Asp Met     370               #   375               #   380 His Val Ala Asp Met Leu Val Lys Val Asn Al #a Leu Ile Lys Asp Arg 385                 3 #90                 3 #95                 4 #00 Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Ar #g Cys Lys Glu Met Ser                 405   #               410   #               415 Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Ty #r Leu Phe Pro Gly His             420       #           425       #           430 Pro Pro Thr         435 <210> SEQ ID NO 10 <211> LENGTH: 835 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(835) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 10 atggagaaca ctgaaaactc agtggattca aaatccatta aaaatttgga ac #caaagatc     60 atacatggaa gcgaatcaat ggactctgga atatccctgg acaacagtta ta #aaatggat    120 tatcctgaga tgggtttatg tataataatt aataataaga attttcataa aa #gcactgga    180 atgacatctc ggtctggtac agatgtcgat gcagcaaacc tcagggaaac at #tcagaaac    240 ttgaaatatg aagtcaggaa taaaaatgat cttacacgtg aagaaattgt gg #aattgatg    300 cgtgatgttt ctaaagaaga tcacagcaaa aggagcagtt ttgtttgtgt gc #ttctgagc    360 catggtgaag aaggaataat ttttggaaca aatggacctg ttgacctgaa aa #aaataaca    420 aactttttca gaggggatcg ttgtagaagt ctaactggaa aacccaaact tt #tcattatt    480 caggcctgcc gtggtacaga actggactgt ggcattgaga cagacagtgg tg #ttgatgat    540 gacatggcgt gtcataaaat accagtggat gccgacttct tgtatgcata ct #ccacagca    600 cctggttatt attcttggcg aaattcaaag gatggctcct ggttcatcca gt #cgctttgt    660 gccatgctga aacagtatgc cgacaagctt gaatttatgc acattcttac cc #gggttaac    720 cgaaaggtgg caacagaatt tgagtccttt tcctttgacg ctacttttca tg #caaagaaa    780 cagattccat gtattgtttc catgctcaca aaagaactct atttttatca ct #aan         835 <210> SEQ ID NO 11 <211> LENGTH: 835 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(835) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 11 tacctcttgt gacttttgag tcacctaagt tttaggtaat ttttaaacct tg #gtttctag     60 tatgtacctt cgcttagtta cctgagacct tatagggacc tgttgtcaat at #tttaccta    120 ataggactct acccaaatac atattattaa ttattattct taaaagtatt tt #cgtgacct    180 tactgtagag ccagaccatg tctacagcta cgtcgtttgg agtccctttg ta #agtctttg    240 aactttatac ttcagtcctt atttttacta gaatgtgcac ttctttaaca cc #ttaactac    300 gcactacaaa gatttcttct agtgtcgttt tcctcgtcaa aacaaacaca cg #aagactcg    360 gtaccacttc ttccttatta aaaaccttgt ttacctggac aactggactt tt #tttattgt    420 ttgaaaaagt ctcccctagc aacatcttca gattgacctt ttgggtttga aa #agtaataa    480 gtccggacgg caccatgtct tgacctgaca ccgtaactct gtctgtcacc ac #aactacta    540 ctgtaccgca cagtatttta tggtcaccta cggctgaaga acatacgtat ga #ggtgtcgt    600 ggaccaataa taagaaccgc tttaagtttc ctaccgagga ccaagtaggt ca #gcgaaaca    660 cggtacgact ttgtcatacg gctgttcgaa cttaaatacg tgtaagaatg gg #cccaattg    720 gctttccacc gttgtcttaa actcaggaaa aggaaactgc gatgaaaagt ac #gtttcttt    780 gtctaaggta cataacaaag gtacgagtgt tttcttgaga taaaaatagt ga #ttn         835 <210> SEQ ID NO 12 <211> LENGTH: 277 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 12 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Ly #s Ser Ile Lys Asn Leu  1               5   #                10   #                15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Me #t Asp Ser Gly Ile Ser             20       #            25       #            30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Gl #u Met Gly Leu Cys Ile         35           #        40           #        45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Th #r Gly Met Thr Ser Arg     50               #    55               #    60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Ar #g Glu Thr Phe Arg Asn 65                   #70                   #75                   #80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Le #u Thr Arg Glu Glu Ile                 85   #                90   #                95 Val Glu Leu Met Arg Asp Val Ser Lys Glu As #p His Ser Lys Arg Ser             100       #           105       #           110 Ser Phe Val Cys Val Leu Leu Ser His Gly Gl #u Glu Gly Ile Ile Phe         115           #       120           #       125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Il #e Thr Asn Phe Phe Arg     130               #   135               #   140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pr #o Lys Leu Phe Ile Ile 145                 1 #50                 1 #55                 1 #60 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gl #y Ile Glu Thr Asp Ser                 165   #               170   #               175 Gly Val Asp Asp Asp Met Ala Cys His Lys Il #e Pro Val Asp Ala Asp             180       #           185       #           190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Ty #r Tyr Ser Trp Arg Asn         195           #       200           #       205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Le #u Cys Ala Met Leu Lys     210               #   215               #   220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Il #e Leu Thr Arg Val Asn 225                 2 #30                 2 #35                 2 #40 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Se #r Phe Asp Ala Thr Phe                 245   #               250   #               255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Se #r Met Leu Thr Lys Glu             260       #           265       #           270 Leu Tyr Phe Tyr His         275 <210> SEQ ID NO 13 <211> LENGTH: 1134 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 13 atggcagaag gcaaccacag aaaaaagcca cttaaggtgt tggaatccct gg #gcaaagat     60 ttcctcactg gtgttttgga taacttggtg gaacaaaatg tactgaactg ga #aggaagag    120 gaaaaaaaga aatattacga tgctaaaact gaagacaaag ttcgggtcat gg #cagactct    180 atgcaagaga agcaacgtat ggcaggacaa atgcttcttc aaaccttttt ta #acatagac    240 caaatatccc ccaataaaaa agctcatccg aatatggagg ctggaccacc tg #agtcagga    300 gaatctacag atgccctcaa gctttgtcct catgaagaat tcctgagact at #gtaaagaa    360 agagctgaag agatctatcc aataaaggag agaaacaacc gcacacgcct gg #ctctcatc    420 atatgcaata cagagtttga ccatctgcct ccgaggaatg gagctgactt tg #acatcaca    480 gggatgaagg agctacttga gggtctggac tatagtgtag atgtagaaga ga #atctgaca    540 gccagggata tggagtcagc gctgagggca tttgctacca gaccagagca ca #agtcctct    600 gacagcacat tcttggtact catgtctcat ggcatcctgg agggaatctg cg #gaactgtg    660 catgatgaga aaaaaccaga tgtgctgctt tatgacacca tcttccagat at #tcaacaac    720 cgcaactgcc tcagtctgaa ggacaaaccc aaggtcatca ttgtccaggc ct #gcagaggt    780 gcaaaccgtg gggaactgtg ggtcagagac tctccagcat ccttggaagt gg #cctcttca    840 cagtcatctg agaacctgga ggaagatgct gtttacaaga cccacgtgga ga #aggacttc    900 attgctttct gctcttcaac gccacacaac gtgtcctgga gagacagcac aa #tgggctct    960 atcttcatca cacaactcat cacatgcttc cagaaatatt cttggtgctg cc #acctagag   1020 gaagtatttc ggaaggtaca gcaatcattt gaaactccaa gggccaaagc tc #aaatgccc   1080 accatagaac gactgtccat gacaagatat ttctacctct ttcctggcaa tt #ga         1134 <210> SEQ ID NO 14 <211> LENGTH: 1134 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 14 taccgtcttc cgttggtgtc ttttttcggt gaattccaca accttaggga cc #cgtttcta     60 aaggagtgac cacaaaacct attgaaccac cttgttttac atgacttgac ct #tccttctc    120 ctttttttct ttataatgct acgattttga cttctgtttc aagcccagta cc #gtctgaga    180 tacgttctct tcgttgcata ccgtcctgtt tacgaagaag tttggaaaaa at #tgtatctg    240 gtttataggg ggttattttt tcgagtaggc ttatacctcc gacctggtgg ac #tcagtcct    300 cttagatgtc tacgggagtt cgaaacagga gtacttctta aggactctga ta #catttctt    360 tctcgacttc tctagatagg ttatttcctc tctttgttgg cgtgtgcgga cc #gagagtag    420 tatacgttat gtctcaaact ggtagacgga ggctccttac ctcgactgaa ac #tgtagtgt    480 ccctacttcc tcgatgaact cccagacctg atatcacatc tacatcttct ct #tagactgt    540 cggtccctat acctcagtcg cgactcccgt aaacgatggt ctggtctcgt gt #tcaggaga    600 ctgtcgtgta agaaccatga gtacagagta ccgtaggacc tcccttagac gc #cttgacac    660 gtactactct tttttggtct acacgacgaa atactgtggt agaaggtcta ta #agttgttg    720 gcgttgacgg agtcagactt cctgtttggg ttccagtagt aacaggtccg ga #cgtctcca    780 cgtttggcac cccttgacac ccagtctctg agaggtcgta ggaaccttca cc #ggagaagt    840 gtcagtagac tcttggacct ccttctacga caaatgttct gggtgcacct ct #tcctgaag    900 taacgaaaga cgagaagttg cggtgtgttg cacaggacct ctctgtcgtg tt #acccgaga    960 tagaagtagt gtgttgagta gtgtacgaag gtctttataa gaaccacgac gg #tggatctc   1020 cttcataaag ccttccatgt cgttagtaaa ctttgaggtt cccggtttcg ag #tttacggg   1080 tggtatcttg ctgacaggta ctgttctata aagatggaga aaggaccgtt aa #ct         1134 <210> SEQ ID NO 15 <211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 15 Met Ala Glu Gly Asn His Arg Lys Lys Pro Le #u Lys Val Leu Glu Ser  1               5   #                10   #                15 Leu Gly Lys Asp Phe Leu Thr Gly Val Leu As #p Asn Leu Val Glu Gln             20       #            25       #            30 Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Ly #s Lys Tyr Tyr Asp Ala         35           #        40           #        45 Lys Thr Glu Asp Lys Val Arg Val Met Ala As #p Ser Met Gln Glu Lys     50               #    55               #    60 Gln Arg Met Ala Gly Gln Met Leu Leu Gln Th #r Phe Phe Asn Ile Asp 65                   #70                   #75                   #80 Gln Ile Ser Pro Asn Lys Lys Ala His Pro As #n Met Glu Ala Gly Pro                 85   #                90   #                95 Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Ly #s Leu Cys Pro His Glu             100       #           105       #           110 Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Gl #u Glu Ile Tyr Pro Ile         115           #       120           #       125 Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Le #u Ile Ile Cys Asn Thr     130               #   135               #   140 Glu Phe Asp His Leu Pro Pro Arg Asn Gly Al #a Asp Phe Asp Ile Thr 145                 1 #50                 1 #55                 1 #60 Gly Met Lys Glu Leu Leu Glu Gly Leu Asp Ty #r Ser Val Asp Val Glu                 165   #               170   #               175 Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Al #a Leu Arg Ala Phe Ala             180       #           185       #           190 Thr Arg Pro Glu His Lys Ser Ser Asp Ser Th #r Phe Leu Val Leu Met         195           #       200           #       205 Ser His Gly Ile Leu Glu Gly Ile Cys Gly Th #r Val His Asp Glu Lys     210               #   215               #   220 Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Ph #e Gln Ile Phe Asn Asn 225                 2 #30                 2 #35                 2 #40 Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Ly #s Val Ile Ile Val Gln                 245   #               250   #               255 Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Tr #p Val Arg Asp Ser Pro             260       #           265       #           270 Ala Ser Leu Glu Val Ala Ser Ser Gln Ser Se #r Glu Asn Leu Glu Glu         275           #       280           #       285 Asp Ala Val Tyr Lys Thr His Val Glu Lys As #p Phe Ile Ala Phe Cys     290               #   295               #   300 Ser Ser Thr Pro His Asn Val Ser Trp Arg As #p Ser Thr Met Gly Ser 305                 3 #10                 3 #15                 3 #20 Ile Phe Ile Thr Gln Leu Ile Thr Cys Phe Gl #n Lys Tyr Ser Trp Cys                 325   #               330   #               335 Cys His Leu Glu Glu Val Phe Arg Lys Val Gl #n Gln Ser Phe Glu Thr             340       #           345       #           350 Pro Arg Ala Lys Ala Gln Met Pro Thr Ile Gl #u Arg Leu Ser Met Thr         355           #       360           #       365 Arg Tyr Phe Tyr Leu Phe Pro Gly Asn     370               #   375 <210> SEQ ID NO 16 <211> LENGTH: 1258 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1258) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 16 atgttcaaag gtatccttca gagtggattg gataacttcg tgataaacca ca #tgctaaag     60 aacaacgtgg ctggacaaac atctatccag accctagtac ctaatacgga tc #aaaagtcg    120 accagtgtaa aaaaagacaa ccacaaaaaa aaaacagtta agatgttgga at #acctgggc    180 aaagatgttc ttcatggtgt ttttaattat ttggcaaaac acgatgttct ga #cattgaag    240 gaagaggaaa agaaaaaata ttatgatgcc aaaattgaag acaaggccct ga #tcttggta    300 gactctttgc gaaagaatcg cgtggctcat caaatgttta cccaaacact tc #tcaatatg    360 gaccaaaaga tcaccagtgt aaaacctctt ctgcaaatcg aggctggacc ac #ctgagtca    420 gcagaatcta caaatatact caaactttgt cctcgtgaag aattcctgag ac #tgtgtaaa    480 aaaaatcatg atgagatcta tccaataaaa aagagagagg accgcagacg cc #tggctctc    540 atcatatgca atacaaagtt tgatcacctg cctgcaagga atggggctca ct #atgacatc    600 gtggggatga aaaggctgct tcaaggcctg ggctacactg tggttgacga aa #agaatctc    660 acagccaggg atatggagtc agtgctgagg gcatttgctg ccagaccaga gc #acaagtcc    720 tctgacagca cgttcttggt actcatgtct catggcatcc tagagggaat ct #gcggaact    780 gcgcataaaa agaaaaaacc ggatgtgctg ctttatgaca ccatcttcca ga #tattcaac    840 aaccgcaact gcctcagtct aaaggacaaa cccaaggtca tcattgtcca gg #cctgcaga    900 ggtgaaaaac atggggaact ctgggtcaga gactctccag catccttggc ag #tcatctct    960 tcacagtcat ctgagaacct ggaggcagat tctgtttgca agatccacga gg #agaaggac   1020 ttcattgctt tctgttcttc aacaccacat aacgtgtcct ggagagaccg ca #caaggggc   1080 tccatcttca ttacggaact catcacatgc ttccagaaat attcttgctg ct #gccaccta   1140 atggaaatat ttcggaaggt acagaaatca tttgaagttc cacaggctaa ag #cccagatg   1200 cccaccatag aacgagcaac cttgacaaga gatttctacc tctttcctgg ca #attgan     1258 <210> SEQ ID NO 17 <211> LENGTH: 1258 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1258) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 17 tacaagtttc cataggaagt ctcacctaac ctattgaagc actatttggt gt #acgatttc     60 ttgttgcacc gacctgtttg tagataggtc tgggatcatg gattatgcct ag #ttttcagc    120 tggtcacatt tttttctgtt ggtgtttttt ttttgtcaat tctacaacct ta #tggacccg    180 tttctacaag aagtaccaca aaaattaata aaccgttttg tgctacaaga ct #gtaacttc    240 cttctccttt tcttttttat aatactacgg ttttaacttc tgttccggga ct #agaaccat    300 ctgagaaacg ctttcttagc gcaccgagta gtttacaaat gggtttgtga ag #agttatac    360 ctggttttct agtggtcaca ttttggagaa gacgtttagc tccgacctgg tg #gactcagt    420 cgtcttagat gtttatatga gtttgaaaca ggagcacttc ttaaggactc tg #acacattt    480 tttttagtac tactctagat aggttatttt ttctctctcc tggcgtctgc gg #accgagag    540 tagtatacgt tatgtttcaa actagtggac ggacgttcct taccccgagt ga #tactgtag    600 cacccctact tttccgacga agttccggac ccgatgtgac accaactgct tt #tcttagag    660 tgtcggtccc tatacctcag tcacgactcc cgtaaacgac ggtctggtct cg #tgttcagg    720 agactgtcgt gcaagaacca tgagtacaga gtaccgtagg atctccctta ga #cgccttga    780 cgcgtatttt tcttttttgg cctacacgac gaaatactgt ggtagaaggt ct #ataagttg    840 ttggcgttga cggagtcaga tttcctgttt gggttccagt agtaacaggt cc #ggacgtct    900 ccactttttg taccccttga gacccagtct ctgagaggtc gtaggaaccg tc #agtagaga    960 agtgtcagta gactcttgga cctccgtcta agacaaacgt tctaggtgct cc #tcttcctg   1020 aagtaacgaa agacaagaag ttgtggtgta ttgcacagga cctctctggc gt #gttccccg   1080 aggtagaagt aatgccttga gtagtgtacg aaggtcttta taagaacgac ga #cggtggat   1140 tacctttata aagccttcca tgtctttagt aaacttcaag gtgtccgatt tc #gggtctac   1200 gggtggtatc ttgctcgttg gaactgttct ctaaagatgg agaaaggacc gt #taactn     1258 <210> SEQ ID NO 18 <211> LENGTH: 418 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 18 Met Phe Lys Gly Ile Leu Gln Ser Gly Leu As #p Asn Phe Val Ile Asn  1               5   #                10   #                15 His Met Leu Lys Asn Asn Val Ala Gly Gln Th #r Ser Ile Gln Thr Leu             20       #            25       #            30 Val Pro Asn Thr Asp Gln Lys Ser Thr Ser Va #l Lys Lys Asp Asn His         35           #        40           #        45 Lys Lys Lys Thr Val Lys Met Leu Glu Tyr Le #u Gly Lys Asp Val Leu     50               #    55               #    60 His Gly Val Phe Asn Tyr Leu Ala Lys His As #p Val Leu Thr Leu Lys 65                   #70                   #75                   #80 Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala Ly #s Ile Glu Asp Lys Ala                 85   #                90   #                95 Leu Ile Leu Val Asp Ser Leu Arg Lys Asn Ar #g Val Ala His Gln Met             100       #           105       #           110 Phe Thr Gln Thr Leu Leu Asn Met Asp Gln Ly #s Ile Thr Ser Val Lys         115           #       120           #       125 Pro Leu Leu Gln Ile Glu Ala Gly Pro Pro Gl #u Ser Ala Glu Ser Thr     130               #   135               #   140 Asn Ile Leu Lys Leu Cys Pro Arg Glu Glu Ph #e Leu Arg Leu Cys Lys 145                 1 #50                 1 #55                 1 #60 Lys Asn His Asp Glu Ile Tyr Pro Ile Lys Ly #s Arg Glu Asp Arg Arg                 165   #               170   #               175 Arg Leu Ala Leu Ile Ile Cys Asn Thr Lys Ph #e Asp His Leu Pro Ala             180       #           185       #           190 Arg Asn Gly Ala His Tyr Asp Ile Val Gly Me #t Lys Arg Leu Leu Gln         195           #       200           #       205 Gly Leu Gly Tyr Thr Val Val Asp Glu Lys As #n Leu Thr Ala Arg Asp     210               #   215               #   220 Met Glu Ser Val Leu Arg Ala Phe Ala Ala Ar #g Pro Glu His Lys Ser 225                 2 #30                 2 #35                 2 #40 Ser Asp Ser Thr Phe Leu Val Leu Met Ser Hi #s Gly Ile Leu Glu Gly                 245   #               250   #               255 Ile Cys Gly Thr Ala His Lys Lys Lys Lys Pr #o Asp Val Leu Leu Tyr             260       #           265       #           270 Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg As #n Cys Leu Ser Leu Lys         275           #       280           #       285 Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cy #s Arg Gly Glu Lys His     290               #   295               #   300 Gly Glu Leu Trp Val Arg Asp Ser Pro Ala Se #r Leu Ala Val Ile Ser 305                 3 #10                 3 #15                 3 #20 Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp Se #r Val Cys Lys Ile His                 325   #               330   #               335 Glu Glu Lys Asp Phe Ile Ala Phe Cys Ser Se #r Thr Pro His Asn Val             340       #           345       #           350 Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Ph #e Ile Thr Glu Leu Ile         355           #       360           #       365 Thr Cys Phe Gln Lys Tyr Ser Cys Cys Cys Hi #s Leu Met Glu Ile Phe     370               #   375               #   380 Arg Lys Val Gln Lys Ser Phe Glu Val Pro Gl #n Ala Lys Ala Gln Met 385                 3 #90                 3 #95                 4 #00 Pro Thr Ile Glu Arg Ala Thr Leu Thr Arg As #p Phe Tyr Leu Phe Pro                 405   #               410   #               415 Gly Asn <210> SEQ ID NO 19 <211> LENGTH: 883 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(883) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 19 atgagctcgg cctcggggct ccgcaggggg cacccggcag gtggggaaga aa #acatgaca     60 gaaacagatg ccttctataa aagagaaatg tttgatccgg cagaaaagta ca #aaatggac    120 cacaggagga gaggaattgc tttaatcttc aatcatgaga ggttcttttg gc #acttaaca    180 ctgccagaaa ggcggggcac ctgcgcagat agagacaatc ttacccgcag gt #tttcagat    240 ctaggatttg aagtgaaatg ctttaatgat cttaaagcag aagaactact gc #tcaaaatt    300 catgaggtgt caactgttag ccacgcagat gccgattgct ttgtgtgtgt ct #tcctgagc    360 catggcgaag gcaatcacat ttatgcatat gatgctaaaa tcgaaattca ga #cattaact    420 ggcttgttca aaggagacaa gtgtcacagc ctggttggaa aacccaagat at #ttatcatc    480 caggcatgtc ggggaaacca gcacgatgtg ccagtcattc ctttggatgt ag #tagataat    540 cagacagaga agttggacac caacataact gaggtggatg cagcctccgt tt #acacgctg    600 cctgctggag ctgacttcct catgtgttac tctgttgcag aaggatatta tt #ctcaccgg    660 gaaactgtga acggctcatg gtacattcaa gatttgtgtg agatgttggg aa #aatatggc    720 tcctccttag agttcacaga actcctcaca ctggtgaaca ggaaagtttc tc #agcgccga    780 gtggactttt gcaaagaccc aagtgcaatt ggaaagaagc aggttccctg tt #ttgcctca    840 atgctaacta aaaagctgca tttctttcca aaatctaatt aan     #                   #883 <210> SEQ ID NO 20 <211> LENGTH: 883 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(883) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 20 tactcgagcc ggagccccga ggcgtccccc gtgggccgtc caccccttct tt #tgtactgt     60 ctttgtctac ggaagatatt ttctctttac aaactaggcc gtcttttcat gt #tttacctg    120 gtgtcctcct ctccttaacg aaattagaag ttagtactct ccaagaaaac cg #tgaattgt    180 gacggtcttt ccgccccgtg gacgcgtcta tctctgttag aatgggcgtc ca #aaagtcta    240 gatcctaaac ttcactttac gaaattacta gaatttcgtc ttcttgatga cg #agttttaa    300 gtactccaca gttgacaatc ggtgcgtcta cggctaacga aacacacaca ga #aggactcg    360 gtaccgcttc cgttagtgta aatacgtata ctacgatttt agctttaagt ct #gtaattga    420 ccgaacaagt ttcctctgtt cacagtgtcg gaccaacctt ttgggttcta ta #aatagtag    480 gtccgtacag cccctttggt cgtgctacac ggtcagtaag gaaacctaca tc #atctatta    540 gtctgtctct tcaacctgtg gttgtattga ctccacctac gtcggaggca aa #tgtgcgac    600 ggacgacctc gactgaagga gtacacaatg agacaacgtc ttcctataat aa #gagtggcc    660 ctttgacact tgccgagtac catgtaagtt ctaaacacac tctacaaccc tt #ttataccg    720 aggaggaatc tcaagtgtct tgaggagtgt gaccacttgt cctttcaaag ag #tcgcggct    780 cacctgaaaa cgtttctggg ttcacgttaa cctttcttcg tccaagggac aa #aacggagt    840 tacgattgat ttttcgacgt aaagaaaggt tttagattaa ttn     #                   #883 <210> SEQ ID NO 21 <211> LENGTH: 293 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 21 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly Hi #s Pro Ala Gly Gly Glu  1               5   #                10   #                15 Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Ly #s Arg Glu Met Phe Asp             20       #            25       #            30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Ar #g Arg Gly Ile Ala Leu         35           #        40           #        45 Ile Phe Asn His Glu Arg Phe Phe Trp His Le #u Thr Leu Pro Glu Arg     50               #    55               #    60 Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Th #r Arg Arg Phe Ser Asp 65                   #70                   #75                   #80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Le #u Lys Ala Glu Glu Leu                 85   #                90   #                95 Leu Leu Lys Ile His Glu Val Ser Thr Val Se #r His Ala Asp Ala Asp             100       #           105       #           110 Cys Phe Val Cys Val Phe Leu Ser His Gly Gl #u Gly Asn His Ile Tyr         115           #       120           #       125 Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Le #u Thr Gly Leu Phe Lys     130               #   135               #   140 Gly Asp Lys Cys His Ser Leu Val Gly Lys Pr #o Lys Ile Phe Ile Ile 145                 1 #50                 1 #55                 1 #60 Gln Ala Cys Arg Gly Asn Gln His Asp Val Pr #o Val Ile Pro Leu Asp                 165   #               170   #               175 Val Val Asp Asn Gln Thr Glu Lys Leu Asp Th #r Asn Ile Thr Glu Val             180       #           185       #           190 Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gl #y Ala Asp Phe Leu Met         195           #       200           #       205 Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser Hi #s Arg Glu Thr Val Asn     210               #   215               #   220 Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Me #t Leu Gly Lys Tyr Gly 225                 2 #30                 2 #35                 2 #40 Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Le #u Val Asn Arg Lys Val                 245   #               250   #               255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pr #o Ser Ala Ile Gly Lys             260       #           265       #           270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Th #r Lys Lys Leu His Phe         275           #       280           #       285 Phe Pro Lys Ser Asn     290 <210> SEQ ID NO 22 <211> LENGTH: 913 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(913) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 22 atggcagatg atcagggctg tattgaagag cagggggttg aggattcagc aa #atgaagat     60 tcagtggatg ctaagccaga ccggtcctcg tttgtaccgt ccctcttcag ta #agaagaag    120 aaaaatgtca ccatgcgatc catcaagacc acccgggacc gagtgcctac at #atcagtac    180 aacatgaatt ttgaaaagct gggcaaatgc atcataataa acaacaagaa ct #ttgataaa    240 gtgacaggta tgggcgttcg aaacggaaca gacaaagatg ccgaggcgct ct #tcaagtgc    300 ttccgaagcc tgggttttga cgtgattgtc tataatgact gctcttgtgc ca #agatgcaa    360 gatctgctta aaaaagcttc tgaagaggac catacaaatg ccgcctgctt cg #cctgcatc    420 ctcttaagcc atggagaaga aaatgtaatt tatgggaaag atggtgtcac ac #caataaag    480 gatttgacag cccactttag gggggataga tgcaaaaccc ttttagagaa ac #ccaaactc    540 ttcttcattc aggcttgccg agggaccgag cttgatgatg gcatccaggc cg #actcgggg    600 cccatcaatg acacagatgc taatcctcga tacaagatcc cagtggaagc tg #acttcctc    660 ttcgcctatt ccacggttcc aggctattac tcgtggagga gcccaggaag ag #gctcctgg    720 tttgtgcaag ccctctgctc catcctggag gagcacggaa aagacctgga aa #tcatgcag    780 atcctcacca gggtgaatga cagagttgcc aggcactttg agtctcagtc tg #atgaccca    840 cacttccatg agaagaagca gatcccctgt gtggtctcca tgctcaccaa gg #aactctac    900 ttcagtcaat agn               #                   #                   #     913 <210> SEQ ID NO 23 <211> LENGTH: 913 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(913) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 23 taccgtctac tagtcccgac ataacttctc gtcccccaac tcctaagtcg tt #tacttcta     60 agtcacctac gattcggtct ggccaggagc aaacatggca gggagaagtc at #tcttcttc    120 tttttacagt ggtacgctag gtagttctgg tgggccctgg ctcacggatg ta #tagtcatg    180 ttgtacttaa aacttttcga cccgtttacg tagtattatt tgttgttctt ga #aactattt    240 cactgtccat acccgcaagc tttgccttgt ctgtttctac ggctccgcga ga #agttcacg    300 aaggcttcgg acccaaaact gcactaacag atattactga cgagaacacg gt #tctacgtt    360 ctagacgaat tttttcgaag acttctcctg gtatgtttac ggcggacgaa gc #ggacgtag    420 gagaattcgg tacctcttct tttacattaa ataccctttc taccacagtg tg #gttatttc    480 ctaaactgtc gggtgaaatc ccccctatct acgttttggg aaaatctctt tg #ggtttgag    540 aagaagtaag tccgaacggc tccctggctc gaactactac cgtaggtccg gc #tgagcccc    600 gggtagttac tgtgtctacg attaggagct atgttctagg gtcaccttcg ac #tgaaggag    660 aagcggataa ggtgccaagg tccgataatg agcacctcct cgggtccttc tc #cgaggacc    720 aaacacgttc gggagacgag gtaggacctc ctcgtgcctt ttctggacct tt #agtacgtc    780 taggagtggt cccacttact gtctcaacgg tccgtgaaac tcagagtcag ac #tactgggt    840 gtgaaggtac tcttcttcgt ctaggggaca caccagaggt acgagtggtt cc #ttgagatg    900 aagtcagtta tcn               #                   #                   #     913 <210> SEQ ID NO 24 <211> LENGTH: 303 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 24 Met Ala Asp Asp Gln Gly Cys Ile Glu Glu Gl #n Gly Val Glu Asp Ser  1               5   #                10   #                15 Ala Asn Glu Asp Ser Val Asp Ala Lys Pro As #p Arg Ser Ser Phe Val             20       #            25       #            30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Va #l Thr Met Arg Ser Ile         35           #        40           #        45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gl #n Tyr Asn Met Asn Phe     50               #    55               #    60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn As #n Lys Asn Phe Asp Lys 65                   #70                   #75                   #80 Val Thr Gly Met Gly Val Arg Asn Gly Thr As #p Lys Asp Ala Glu Ala                 85   #                90   #                95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe As #p Val Ile Val Tyr Asn             100       #           105       #           110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Le #u Lys Lys Ala Ser Glu         115           #       120           #       125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cy #s Ile Leu Leu Ser His     130               #   135               #   140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gl #y Val Thr Pro Ile Lys 145                 1 #50                 1 #55                 1 #60 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cy #s Lys Thr Leu Leu Glu                 165   #               170   #               175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Ar #g Gly Thr Glu Leu Asp             180       #           185       #           190 Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile As #n Asp Thr Asp Ala Asn         195           #       200           #       205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Ph #e Leu Phe Ala Tyr Ser     210               #   215               #   220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pr #o Gly Arg Gly Ser Trp 225                 2 #30                 2 #35                 2 #40 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Gl #u His Gly Lys Asp Leu                 245   #               250   #               255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn As #p Arg Val Ala Arg His             260       #           265       #           270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe Hi #s Glu Lys Lys Gln Ile         275           #       280           #       285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Le #u Tyr Phe Ser Gln     290               #   295               #   300 <210> SEQ ID NO 25 <211> LENGTH: 1493 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1493) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 25 atggacttca gcagaaatct ttatgatatt ggggaacaac tggacagtga ag #atctggcc     60 tccctcaagt tcctgagcct ggactacatt ccgcaaagga agcaagaacc ca #tcaaggat    120 gccttgatgt tattccagag actccaggaa aagagaatgt tggaggaaag ca #atctgtcc    180 ttcctgaagg agctgctctt ccgaattaat agactggatt tgctgattac ct #acctaaac    240 actagaaagg aggagatgga aagggaactt cagacaccag gcagggctca aa #tttctgcc    300 tacaggttcc acttctgccg catgagctgg gctgaagcaa acagccagtg cc #agacacag    360 tctgtacctt tctggcggag ggtcgatcat ctattaataa gggtcatgct ct #atcagatt    420 tcagaagaag tgagcagatc agaattgagg tcttttaagt ttcttttgca ag #aggaaatc    480 tccaaatgca aactggatga tgacatgaac ctgctggata ttttcataga ga #tggagaag    540 agggtcatcc tgggagaagg aaagttggac atcctgaaaa gagtctgtgc cc #aaatcaac    600 aagagcctgc tgaagataat caacgactat gaagaattca gcaaagggga gg #agttgtgt    660 ggggtaatga caatctcgga ctctccaaga gaacaggata gtgaatcaca ga #ctttggac    720 aaagtttacc aaatgaaaag caaacctcgg ggatactgtc tgatcatcaa ca #atcacaat    780 tttgcaaaag cacgggagaa agtgcccaaa cttcacagca ttagggacag ga #atggaaca    840 cacttggatg caggggcttt gaccacgacc tttgaagagc ttcattttga ga #tcaagccc    900 caccatgact gcacagtaga gcaaatctat gagattttga aaatctacca ac #tcatggac    960 cacagtaaca tggactgctt catctgctgt atcctctccc atggagacaa gg #gcatcatc   1020 tatggcactg atggacagga ggcccccatc tatgagctga catctcagtt ca #ctggtttg   1080 aagtgccctt cccttgctgg aaaacccaaa gtgtttttta ttcaggcttg tc #agggggat   1140 aactaccaga aaggtatacc tgttgagact gattcagagg agcaacccta tt #tagaaatg   1200 gatttatcat cacctcaaac gagatatatc ccggatgagg ctgactttct gc #tggggatg   1260 gccactgtga ataactgtgt ttcctaccga aaccctgcag agggaacctg gt #acatccag   1320 tcactttgcc agagcctgag agagcgatgt cctcgaggcg atgatattct ca #ccatcctg   1380 actgaagtga actatgaagt aagcaacaag gatgacaaga aaaacatggg ga #aacagatg   1440 cctcagccta ctttcacact aagaaaaaaa cttgtcttcc cttctgattg an #n          1493 <210> SEQ ID NO 26 <211> LENGTH: 1493 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1493) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 26 tacctgaagt cgtctttaga aatactataa ccccttgttg acctgtcact tc #tagaccgg     60 agggagttca aggactcgga cctgatgtaa ggcgtttcct tcgttcttgg gt #agttccta    120 cggaactaca ataaggtctc tgaggtcctt ttctcttaca acctcctttc gt #tagacagg    180 aaggacttcc tcgacgagaa ggcttaatta tctgacctaa acgactaatg ga #tggatttg    240 tgatctttcc tcctctacct ttcccttgaa gtctgtggtc cgtcccgagt tt #aaagacgg    300 atgtccaagg tgaagacggc gtactcgacc cgacttcgtt tgtcggtcac gg #tctgtgtc    360 agacatggaa agaccgcctc ccagctagta gataattatt cccagtacga ga #tagtctaa    420 agtcttcttc actcgtctag tcttaactcc agaaaattca aagaaaacgt tc #tcctttag    480 aggtttacgt ttgacctact actgtacttg gacgacctat aaaagtatct ct #acctcttc    540 tcccagtagg accctcttcc tttcaacctg taggactttt ctcagacacg gg #tttagttg    600 ttctcggacg acttctatta gttgctgata cttcttaagt cgtttcccct cc #tcaacaca    660 ccccattact gttagagcct gagaggttct cttgtcctat cacttagtgt ct #gaaacctg    720 tttcaaatgg tttacttttc gtttggagcc cctatgacag actagtagtt gt #tagtgtta    780 aaacgttttc gtgccctctt tcacgggttt gaagtgtcgt aatccctgtc ct #taccttgt    840 gtgaacctac gtccccgaaa ctggtgctgg aaacttctcg aagtaaaact ct #agttcggg    900 gtggtactga cgtgtcatct cgtttagata ctctaaaact tttagatggt tg #agtacctg    960 gtgtcattgt acctgacgaa gtagacgaca taggagaggg tacctctgtt cc #cgtagtag   1020 ataccgtgac tacctgtcct ccgggggtag atactcgact gtagagtcaa gt #gaccaaac   1080 ttcacgggaa gggaacgacc ttttgggttt cacaaaaaat aagtccgaac ag #tcccccta   1140 ttgatggtct ttccatatgg acaactctga ctaagtctcc tcgttgggat aa #atctttac   1200 ctaaatagta gtggagtttg ctctatatag ggcctactcc gactgaaaga cg #acccctac   1260 cggtgacact tattgacaca aaggatggct ttgggacgtc tcccttggac ca #tgtaggtc   1320 agtgaaacgg tctcggactc tctcgctaca ggagctccgc tactataaga gt #ggtaggac   1380 tgacttcact tgatacttca ttcgttgttc ctactgttct ttttgtaccc ct #ttgtctac   1440 ggagtcggat gaaagtgtga ttcttttttt gaacagaagg gaagactaac tn #n          1493 <210> SEQ ID NO 27 <211> LENGTH: 476 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 27 Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl #y Glu Gln Leu Asp Ser  1               5   #                10   #                15 Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le #u Asp Tyr Ile Pro Gln             20       #            25       #            30 Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me #t Leu Phe Gln Arg Leu         35           #        40           #        45 Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le #u Ser Phe Leu Lys Glu     50               #    55               #    60 Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Le #u Ile Thr Tyr Leu Asn 65                   #70                   #75                   #80 Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gl #n Thr Pro Gly Arg Ala                 85   #                90   #                95 Gln Ile Ser Ala Tyr Arg Phe His Phe Cys Ar #g Met Ser Trp Ala Glu             100       #           105       #           110 Ala Asn Ser Gln Cys Gln Thr Gln Ser Val Pr #o Phe Trp Arg Arg Val         115           #       120           #       125 Asp His Leu Leu Ile Arg Val Met Leu Tyr Gl #n Ile Ser Glu Glu Val     130               #   135               #   140 Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Le #u Leu Gln Glu Glu Ile 145                 1 #50                 1 #55                 1 #60 Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Le #u Leu Asp Ile Phe Ile                 165   #               170   #               175 Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gl #y Lys Leu Asp Ile Leu             180       #           185       #           190 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Le #u Leu Lys Ile Ile Asn         195           #       200           #       205 Asp Tyr Glu Glu Phe Ser Lys Gly Glu Glu Le #u Cys Lys Val Tyr Gln     210               #   215               #   220 Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Il #e Ile Asn Asn His Asn 225                 2 #30                 2 #35                 2 #40 Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Le #u His Ser Ile Arg Asp                 245   #               250   #               255 Arg Asn Gly Thr His Leu Asp Ala Gly Ala Le #u Thr Thr Thr Phe Glu             260       #           265       #           270 Glu Leu His Phe Glu Ile Lys Pro His His As #p Cys Thr Val Glu Gln         275           #       280           #       285 Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu Me #t Asp His Ser Asn Met     290               #   295               #   300 Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gl #y Asp Lys Gly Ile Ile 305                 3 #10                 3 #15                 3 #20 Tyr Gly Thr Asp Gly Gln Glu Ala Pro Ile Ty #r Glu Leu Thr Ser Gln                 325   #               330   #               335 Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gl #y Lys Pro Lys Val Phe             340       #           345       #           350 Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gl #n Lys Gly Ile Pro Val         355           #       360           #       365 Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Gl #u Met Asp Leu Ser Ser     370               #   375               #   380 Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala As #p Phe Leu Leu Gly Met 385                 3 #90                 3 #95                 4 #00 Ala Thr Val Asn Asn Cys Val Ser Tyr Arg As #n Pro Ala Glu Gly Thr                 405   #               410   #               415 Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Ar #g Glu Arg Cys Pro Arg             420       #           425       #           430 Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Va #l Asn Tyr Glu Val Ser         435           #       440           #       445 Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gl #n Met Pro Gln Pro Thr     450               #   455               #   460 Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Se #r Asp 465                 4 #70                 4 #75 <210> SEQ ID NO 28 <211> LENGTH: 1252 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1252) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 28 atggacgaag cggatcggcg gctcctgcgg cggtgccggc tgcggctggt gg #aagagctg     60 caggtggacc agctctggga cgccctgctg agcagcgagc tgttcaggcc cc #atatgatc    120 gaggacatcc agcgggcagg ctctggatct cggcgggatc aggccaggca gc #tgatcata    180 gatctggaga ctcgagggag tcaggctctt cctttgttca tctcctgctt ag #aggacaca    240 ggccaggaca tgctggcttc gtttctgcga actaacaggc aagcagcaaa gt #tgtcgaag    300 ccaaccctag aaaaccttac cccagtggtg ctcagaccag agattcgcaa ac #cagaggtt    360 ctcagaccgg aaacacccag accagtggac attggttctg gaggatttgg tg #atgtcggt    420 gctcttgaga gtttgagggg aaatgcagat ttggcttaca tcctgagcat gg #agccctgt    480 ggccactgcc tcattatcaa caatgtgaac ttctgccgtg agtccgggct cc #gcacccgc    540 actggctcca acatcgactg tgagaagttg cggcgtcgct tctcctcgcc gc #atttcatg    600 gtggaggtga agggcgacct gactgccaag aaaatggtgc tggctttgct gg #agctggcg    660 cggcaggacc acggtgctct ggactgctgc gtggtggtca ttctctctca cg #gctgtcag    720 gccagccacc tgcagttccc aggggctgtc tacggcacag atggatgccc tg #tgtcggtc    780 gagaagattg tgaacatctt caatgggacc agctgcccca gcctgggagg aa #agcccaag    840 ctctttttca tccaggcctg tggtggggag cagaaagacc atgggtttga gg #tggcctcc    900 acttcccctg aagacgagtc ccctggcagt aaccccgagc cagatgccac cc #cgttccag    960 gaaggtttga ggaccttcga ccagctggac gccatatcta gtttgcccac ac #ccagtgac   1020 atctttgtgt cctactctac tttcccaggt tttgtttcct ggagggaccc ca #agagtggc   1080 tcctggtacg ttgagaccct ggacgacatc tttgagcagt gggctcactc tg #aagacctg   1140 cagtccctcc tgcttagggt cgctaatgct gtttcggtga aagggattta ta #aacagatg   1200 cctggttgct ttaatttcct ccggaaaaaa cttttcttta aaacatcata an #           1252 <210> SEQ ID NO 29 <211> LENGTH: 1252 <212> TYPE: DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1252) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 29 tacctgcttc gcctagccgc cgaggacgcc gccacggccg acgccgacca cc #ttctcgac     60 gtccacctgg tcgagaccct gcgggacgac tcgtcgctcg acaagtccgg gg #tatactag    120 ctcctgtagg tcgcccgtcc gagacctaga gccgccctag tccggtccgt cg #actagtat    180 ctagacctct gagctccctc agtccgagaa ggaaacaagt agaggacgaa tc #tcctgtgt    240 ccggtcctgt acgaccgaag caaagacgct tgattgtccg ttcgtcgttt ca #acagcttc    300 ggttgggatc ttttggaatg gggtcaccac gagtctggtc tctaagcgtt tg #gtctccaa    360 gagtctggcc tttgtgggtc tggtcacctg taaccaagac ctcctaaacc ac #tacagcca    420 cgagaactct caaactcccc tttacgtcta aaccgaatgt aggactcgta cc #tcgggaca    480 ccggtgacgg agtaatagtt gttacacttg aagacggcac tcaggcccga gg #cgtgggcg    540 tgaccgaggt tgtagctgac actcttcaac gccgcagcga agaggagcgg cg #taaagtac    600 cacctccact tcccgctgga ctgacggttc ttttaccacg accgaaacga cc #tcgaccgc    660 gccgtcctgg tgccacgaga cctgacgacg caccaccagt aagagagagt gc #cgacagtc    720 cggtcggtgg acgtcaaggg tccccgacag atgccgtgtc tacctacggg ac #acagccag    780 ctcttctaac acttgtagaa gttaccctgg tcgacggggt cggaccctcc tt #tcgggttc    840 gagaaaaagt aggtccggac accacccctc gtctttctgg tacccaaact cc #accggagg    900 tgaaggggac ttctgctcag gggaccgtca ttggggctcg gtctacggtg gg #gcaaggtc    960 cttccaaact cctggaagct ggtcgacctg cggtatagat caaacgggtg tg #ggtcactg   1020 tagaaacaca ggatgagatg aaagggtcca aaacaaagga cctccctggg gt #tctcaccg   1080 aggaccatgc aactctggga cctgctgtag aaactcgtca cccgagtgag ac #ttctggac   1140 gtcagggagg acgaatccca gcgattacga caaagccact ttccctaaat at #ttgtctac   1200 ggaccaacga aattaaagga ggcctttttt gaaaagaaat tttgtagtat tn #           1252 <210> SEQ ID NO 30 <211> LENGTH: 416 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 30 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Ar #g Cys Arg Leu Arg Leu  1               5   #                10   #                15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp As #p Ala Leu Leu Ser Ser             20       #            25       #            30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Il #e Gln Arg Ala Gly Ser         35           #        40           #        45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Il #e Ile Asp Leu Glu Thr     50               #    55               #    60 Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Se #r Cys Leu Glu Asp Thr 65                   #70                   #75                   #80 Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Th #r Asn Arg Gln Ala Ala                 85   #                90   #                95 Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Th #r Pro Val Val Leu Arg             100       #           105       #           110 Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pr #o Glu Thr Pro Arg Pro         115           #       120           #       125 Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Va #l Gly Ala Leu Glu Ser     130               #   135               #   140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Le #u Ser Met Glu Pro Cys 145                 1 #50                 1 #55                 1 #60 Gly His Cys Leu Ile Ile Asn Asn Val Asn Ph #e Cys Arg Glu Ser Gly                 165   #               170   #               175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cy #s Glu Lys Leu Arg Arg             180       #           185       #           190 Arg Phe Ser Ser Pro His Phe Met Val Glu Va #l Lys Gly Asp Leu Thr         195           #       200           #       205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Le #u Ala Arg Gln Asp His     210               #   215               #   220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Le #u Ser His Gly Cys Gln 225                 2 #30                 2 #35                 2 #40 Ala Ser His Leu Gln Phe Pro Gly Ala Val Ty #r Gly Thr Asp Gly Cys                 245   #               250   #               255 Pro Val Ser Val Glu Lys Ile Val Asn Ile Ph #e Asn Gly Thr Ser Cys             260       #           265       #           270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Ph #e Ile Gln Ala Cys Gly         275           #       280           #       285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Al #a Ser Thr Ser Pro Glu     290               #   295               #   300 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro As #p Ala Thr Pro Phe Gln 305                 3 #10                 3 #15                 3 #20 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Al #a Ile Ser Ser Leu Pro                 325   #               330   #               335 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Th #r Phe Pro Gly Phe Val             340       #           345       #           350 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Ty #r Val Glu Thr Leu Asp         355           #       360           #       365 Asp Ile Phe Glu Gln Trp Ala His Ser Glu As #p Leu Gln Ser Leu Leu     370               #   375               #   380 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gl #y Ile Tyr Lys Gln Met 385                 3 #90                 3 #95                 4 #00 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Le #u Phe Phe Lys Thr Ser                 405   #               410   #               415 <210> SEQ ID NO 31 <211> LENGTH: 1440 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 31 atgaaatctc aaggtcaaca ttggtattcc agttcagata aaaactgtaa ag #tgagcttt     60 cgtgagaagc ttctgattat tgattcaaac ctgggggtcc aagatgtgga ga #acctcaag    120 tttctctgca taggattggt ccccaacaag aagctggaga agtccagctc ag #cctcagat    180 gtttttgaac atctcttggc agaggatctg ctgagtgagg aagacccttt ct #tcctggca    240 gaactcctct atatcatacg gcagaagaag ctgctgcagc acctcaactg ta #ccaaagag    300 gaagtggagc gactgctgcc cacccgacaa agggtttctc tgtttagaaa cc #tgctctac    360 gaactgtcag aaggcattga ctcagagaac ttaaaggaca tgatcttcct tc #tgaaagac    420 tcgcttccca aaactgaaat gacctcccta agtttcctgg catttctaga ga #aacaaggt    480 aaaatagatg aagataatct gacatgcctg gaggacctct gcaaaacagt tg #tacctaaa    540 cttttgagaa acatagagaa atacaaaaga gagaaagcta tccagatagt ga #cacctcct    600 gtagacaagg aagccgagtc gtatcaagga gaggaagaac tagtttccca aa #cagatgtt    660 aagacattct tggaagcctt accgagggca gctgtgtaca ggatgaatcg ga #accacaga    720 ggcctctgtg tcattgtcaa caaccacagc tttacctccc tgaaggacag ac #aaggaacc    780 cataaagatg ctgagatcct gagtcatgtg ttccagtggc ttgggttcac ag #tgcatata    840 cacaataatg tgacgaaagt ggaaatggag atggtcctgc agaagcagaa gt #gcaatcca    900 gcccatgccg acggggactg cttcgtgttc tgtattctga cccatgggag at #ttggagct    960 gtctactctt cggatgaggc cctcattccc attcgggaga tcatgtctca ct #tcacagcc   1020 ctgcagtgcc ctagactggc tgaaaaacct aaactctttt tcatccaggc ct #gccaaggt   1080 gaagagatac agccttccgt atccatcgaa gcagatgctc tgaaccctga gc #aggcaccc   1140 acttccctgc aggacagtat tcctgccgag gctgacttcc tacttggtct gg #ccactgtc   1200 ccaggctatg tatcctttcg gcatgtggag gaaggcagct ggtatattca gt #ctctgtgt   1260 aatcatctga agaaattggt cccaagacat gaagacatct tatccatcct ca #ctgctgtc   1320 aacgatgatg tgagtcgaag agtggacaaa cagggaacaa agaaacagat gc #cccagcct   1380 gctttcacac taaggaaaaa actagtattc cctgtgcccc tggatgcact tt #caatatag   1440 <210> SEQ ID NO 32 <211> LENGTH: 1440 <212> TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 32 tactttagag ttccagttgt aaccataagg tcaagtctat ttttgacatt tc #actcgaaa     60 gcactcttcg aagactaata actaagtttg gacccccagg ttctacacct ct #tggagttc    120 aaagagacgt atcctaacca ggggttgttc ttcgacctct tcaggtcgag tc #ggagtcta    180 caaaaacttg tagagaaccg tctcctagac gactcactcc ttctgggaaa ga #aggaccgt    240 cttgaggaga tatagtatgc cgtcttcttc gacgacgtcg tggagttgac at #ggtttctc    300 cttcacctcg ctgacgacgg gtgggctgtt tcccaaagag acaaatcttt gg #acgagatg    360 cttgacagtc ttccgtaact gagtctcttg aatttcctgt actagaagga ag #actttctg    420 agcgaagggt tttgacttta ctggagggat tcaaaggacc gtaaagatct ct #ttgttcca    480 ttttatctac ttctattaga ctgtacggac ctcctggaga cgttttgtca ac #atggattt    540 gaaaactctt tgtatctctt tatgttttct ctctttcgat aggtctatca ct #gtggagga    600 catctgttcc ttcggctcag catagttcct ctccttcttg atcaaagggt tt #gtctacaa    660 ttctgtaaga accttcggaa tggctcccgt cgacacatgt cctacttagc ct #tggtgtct    720 ccggagacac agtaacagtt gttggtgtcg aaatggaggg acttcctgtc tg #ttccttgg    780 gtatttctac gactctagga ctcagtacac aaggtcaccg aacccaagtg tc #acgtatat    840 gtgttattac actgctttca cctttacctc taccaggacg tcttcgtctt ca #cgttaggt    900 cgggtacggc tgcccctgac gaagcacaag acataagact gggtaccctc ta #aacctcga    960 cagatgagaa gcctactccg ggagtaaggg taagccctct agtacagagt ga #agtgtcgg   1020 gacgtcacgg gatctgaccg actttttgga tttgagaaaa agtaggtccg ga #cggttcca   1080 cttctctatg tcggaaggca taggtagctt cgtctacgag acttgggact cg #tccgtggg   1140 tgaagggacg tcctgtcata aggacggctc cgactgaagg atgaaccaga cc #ggtgacag   1200 ggtccgatac ataggaaagc cgtacacctc cttccgtcga ccatataagt ca #gagacaca   1260 ttagtagact tctttaacca gggttctgta cttctgtaga ataggtagga gt #gacgacag   1320 ttgctactac actcagcttc tcacctgttt gtcccttgtt tctttgtcta cg #gggtcgga   1380 cgaaagtgtg attccttttt tgatcataag ggacacgggg acctacgtga aa #gttatatc   1440 <210> SEQ ID NO 33 <211> LENGTH: 479 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 33 Met Lys Ser Gln Gly Gln His Trp Tyr Ser Se #r Ser Asp Lys Asn Cys  1               5   #                10   #                15 Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Il #e Asp Ser Asn Leu Gly             20       #            25       #            30 Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cy #s Ile Gly Leu Val Pro         35           #        40           #        45 Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Se #r Asp Val Phe Glu His     50               #    55               #    60 Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu As #p Pro Phe Phe Leu Ala 65                   #70                   #75                   #80 Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Le #u Leu Gln His Leu Asn                 85   #                90   #                95 Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pr #o Thr Arg Gln Arg Val             100       #           105       #           110 Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Se #r Glu Gly Ile Asp Ser         115           #       120           #       125 Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Ly #s Asp Ser Leu Pro Lys     130               #   135               #   140 Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Ph #e Leu Glu Lys Gln Gly 145                 1 #50                 1 #55                 1 #60 Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Gl #u Asp Leu Cys Lys Thr                 165   #               170   #               175 Val Val Pro Lys Leu Leu Arg Asn Ile Glu Ly #s Tyr Lys Arg Glu Lys             180       #           185       #           190 Ala Ile Gln Ile Val Thr Pro Pro Val Asp Ly #s Glu Ala Glu Ser Tyr         195           #       200           #       205 Gln Gly Glu Glu Glu Leu Val Ser Gln Thr As #p Val Lys Thr Phe Leu     210               #   215               #   220 Glu Ala Leu Pro Arg Ala Ala Val Tyr Arg Me #t Asn Arg Asn His Arg 225                 2 #30                 2 #35                 2 #40 Gly Leu Cys Val Ile Val Asn Asn His Ser Ph #e Thr Ser Leu Lys Asp                 245   #               250   #               255 Arg Gln Gly Thr His Lys Asp Ala Glu Ile Le #u Ser His Val Phe Gln             260       #           265       #           270 Trp Leu Gly Phe Thr Val His Ile His Asn As #n Val Thr Lys Val Glu         275           #       280           #       285 Met Glu Met Val Leu Gln Lys Gln Lys Cys As #n Pro Ala His Ala Asp     290               #   295               #   300 Gly Asp Cys Phe Val Phe Cys Ile Leu Thr Hi #s Gly Arg Phe Gly Ala 305                 3 #10                 3 #15                 3 #20 Val Tyr Ser Ser Asp Glu Ala Leu Ile Pro Il #e Arg Glu Ile Met Ser                 325   #               330   #               335 His Phe Thr Ala Leu Gln Cys Pro Arg Leu Al #a Glu Lys Pro Lys Leu             340       #           345       #           350 Phe Phe Ile Gln Ala Cys Gln Gly Glu Glu Il #e Gln Pro Ser Val Ser         355           #       360           #       365 Ile Glu Ala Asp Ala Leu Asn Pro Glu Gln Al #a Pro Thr Ser Leu Gln     370               #   375               #   380 Asp Ser Ile Pro Ala Glu Ala Asp Phe Leu Le #u Gly Leu Ala Thr Val 385                 3 #90                 3 #95                 4 #00 Pro Gly Tyr Val Ser Phe Arg His Val Glu Gl #u Gly Ser Trp Tyr Ile                 405   #               410   #               415 Gln Ser Leu Cys Asn His Leu Lys Lys Leu Va #l Pro Arg His Glu Asp             420       #           425       #           430 Ile Leu Ser Ile Leu Thr Ala Val Asn Asp As #p Val Ser Arg Arg Val         435           #       440           #       445 Asp Lys Gln Gly Thr Lys Lys Gln Met Pro Gl #n Pro Ala Phe Thr Leu     450               #   455               #   460 Arg Lys Lys Leu Val Phe Pro Val Pro Leu As #p Ala Leu Ser Ile 465                 4 #70                 4 #75 <210> SEQ ID NO 34 <211> LENGTH: 290 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Rev-Caspase-3 contructed fro #m human caspase -3 <400> SEQUENCE: 34 Met Ile Glu Thr Asp Ser Gly Val Asp Asp As #p Met Ala Cys His Lys  1               5   #                10   #                15 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Ty #r Ser Thr Ala Pro Gly             20       #            25       #            30 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Se #r Trp Phe Ile Gln Ser         35           #        40           #        45 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Ly #s Leu Glu Phe Met His     50               #    55               #    60 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Th #r Glu Phe Glu Ser Phe 65                   #70                   #75                   #80 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gl #n Ile Pro Cys Ile Val                 85   #                90   #                95 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr Hi #s Asp Glu Val Asp Gly             100       #           105       #           110 Gly Ser Pro Met Glu Asn Thr Glu Asn Ser Va #l Asp Ser Lys Ser Ile         115           #       120           #       125 Lys Asn Leu Glu Pro Lys Ile Ile His Gly Se #r Glu Ser Met Asp Ser     130               #   135               #   140 Gly Ile Ser Leu Asp Asn Ser Tyr Lys Met As #p Tyr Pro Glu Met Gly 145                 1 #50                 1 #55                 1 #60 Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe Hi #s Lys Ser Thr Gly Met                 165   #               170   #               175 Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Al #a Asn Leu Arg Glu Thr             180       #           185       #           190 Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Ly #s Asn Asp Leu Thr Arg         195           #       200           #       205 Glu Glu Ile Val Glu Leu Met Arg Asp Val Se #r Lys Glu Asp His Ser     210               #   215               #   220 Lys Arg Ser Ser Phe Val Cys Val Leu Leu Se #r His Gly Glu Glu Gly 225                 2 #30                 2 #35                 2 #40 Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Le #u Lys Lys Ile Thr Asn                 245   #               250   #               255 Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Th #r Gly Lys Pro Lys Leu             260       #           265       #           270 Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Le #u Asp Cys Gly Ile Glu         275           #       280           #       285 Thr Asp     290 <210> SEQ ID NO 35 <211> LENGTH: 285 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Uncleavable Rev-Caspase-3 co #ntructed from human       caspase -3 <400> SEQUENCE: 35 Met Ile Glu Thr Asp Ser Gly Val Asp Asp As #p Met Ala Cys His Lys  1               5   #                10   #                15 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Ty #r Ser Thr Ala Pro Gly             20       #            25       #            30 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Se #r Trp Phe Ile Gln Ser         35           #        40           #        45 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Ly #s Leu Glu Phe Met His     50               #    55               #    60 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Th #r Glu Phe Glu Ser Phe 65                   #70                   #75                   #80 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gl #n Ile Pro Cys Ile Val                 85   #                90   #                95 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr Hi #s Gly Ser Pro Met Glu             100       #           105       #           110 Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Il #e Lys Asn Leu Glu Pro         115           #       120           #       125 Lys Ile Ile His Gly Ser Glu Ser Met Ala Se #r Gly Ile Ser Leu Asp     130               #   135               #   140 Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gl #y Leu Cys Ile Ile Ile 145                 1 #50                 1 #55                 1 #60 Asn Asn Lys Asn Phe His Lys Ser Thr Gly Me #t Thr Ser Arg Ser Gly                 165   #               170   #               175 Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Th #r Phe Arg Asn Leu Lys             180       #           185       #           190 Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Ar #g Glu Glu Ile Val Glu         195           #       200           #       205 Leu Met Arg Asp Val Ser Lys Glu Asp His Se #r Lys Arg Ser Ser Phe     210               #   215               #   220 Val Cys Val Leu Leu Ser His Gly Glu Glu Gl #y Ile Ile Phe Gly Thr 225                 2 #30                 2 #35                 2 #40 Asn Gly Pro Val Asp Leu Lys Lys Ile Thr As #n Phe Phe Arg Gly Asp                 245   #               250   #               255 Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Le #u Phe Ile Ile Gln Ala             260       #           265       #           270 Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Gl #u Thr Asp         275           #       280           #       285 <210> SEQ ID NO 36 <211> LENGTH: 300 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Rev-Caspase-6 contructed fro #m human caspase-6 <400> SEQUENCE: 36 Met Val Glu Ile Asp Ala Ala Ser Val Tyr Th #r Leu Pro Ala Gly Ala  1               5   #                10   #                15 Asp Phe Leu Met Cys Tyr Ser Val Ala Glu Gl #y Tyr Tyr Ser His Arg             20       #            25       #            30 Glu Thr Val Asn Gly Ser Trp Tyr Ile Gln As #p Leu Cys Glu Met Leu         35           #        40           #        45 Gly Lys Tyr Gly Ser Ser Leu Glu Phe Thr Gl #u Leu Leu Thr Leu Val     50               #    55               #    60 Asn Arg Lys Val Ser Gln Arg Arg Val Asp Ph #e Cys Lys Asp Pro Ser 65                   #70                   #75                   #80 Ala Ile Gly Lys Lys Gln Val Pro Cys Phe Al #a Ser Met Leu Thr Lys                 85   #                90   #                95 Lys Leu His Phe Phe Pro Lys Ser Asn Leu Gl #u His His His His His             100       #           105       #           110 His Val Glu Ile Asp Gly Gly Ser Pro Met Se #r Ser Ala Ser Gly Leu         115           #       120           #       125 Arg Arg Gly His Pro Ala Gly Gly Glu Glu As #n Met Thr Glu Thr Asp     130               #   135               #   140 Ala Phe Tyr Lys Arg Glu Met Phe Asp Pro Al #a Glu Lys Tyr Lys Met 145                 1 #50                 1 #55                 1 #60 Asp His Arg Arg Arg Gly Ile Ala Leu Ile Ph #e Asn His Glu Arg Phe                 165   #               170   #               175 Phe Trp His Leu Thr Leu Pro Glu Arg Arg Gl #y Thr Cys Ala Asp Arg             180       #           185       #           190 Asp Asn Leu Thr Arg Arg Phe Ser Asp Leu Gl #y Phe Glu Val Lys Cys         195           #       200           #       205 Phe Asn Asp Leu Lys Ala Glu Glu Leu Leu Le #u Lys Ile His Glu Val     210               #   215               #   220 Ser Thr Val Ser His Ala Asp Ala Asp Cys Ph #e Val Cys Val Phe Leu 225                 2 #30                 2 #35                 2 #40 Ser His Gly Glu Gly Asn His Ile Tyr Ala Ty #r Asp Ala Lys Ile Glu                 245   #               250   #               255 Ile Gln Thr Leu Thr Gly Leu Phe Lys Gly As #p Lys Cys His Ser Leu             260       #           265       #           270 Val Gly Lys Pro Lys Ile Phe Ile Ile Gln Al #a Cys Arg Gly Asn Gln         275           #       280           #       285 His Asp Val Pro Val Ile Pro Leu Asp Val Va #l Asp     290               #   295               #   300 <210> SEQ ID NO 37 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(5) <223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 37 Asp Xaa Xaa Asp Gly  1               5 <210> SEQ ID NO 38 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <400> SEQUENCE: 38 Asp Glu Val Asp Gly  1               5 <210> SEQ ID NO 39 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <400> SEQUENCE: 39 Ile Glu Thr Asp Gly  1               5 <210> SEQ ID NO 40 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <400> SEQUENCE: 40 Tyr Val Ala Asp Gly  1               5 <210> SEQ ID NO 41 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <400> SEQUENCE: 41 Tyr Val His Asp Gly  1               5 <210> SEQ ID NO 42 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <400> SEQUENCE: 42 Trp Glu His Asp Gly  1               5 <210> SEQ ID NO 43 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for large s #ubunit of caspase-3 <400> SEQUENCE: 43 atggagaaca ctgaaaactc ag            #                   #                 22 <210> SEQ ID NO 44 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for large s #ubunit of caspase-3 <400> SEQUENCE: 44 gtcatcatca acacctcagt ct            #                   #                 22 <210> SEQ ID NO 45 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for small s #ubunit of caspase-3 <400> SEQUENCE: 45 ggatccatga ttgagacaga cagtgg           #                   #              26 <210> SEQ ID NO 46 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for small s #ubunit of caspase-3 <400> SEQUENCE: 46 atcaacttca tcgtgataaa aatagagttc          #                   #           30 <210> SEQ ID NO 47 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for large s #ubunit of caspase-6 <400> SEQUENCE: 47 atgagctcgg cctcgggg              #                   #                   #  18 <210> SEQ ID NO 48 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for large s #ubunit of caspase-6 <400> SEQUENCE: 48 ttaatctact acatccaaag g            #                   #                   #21 <210> SEQ ID NO 49 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for small s #ubunit of caspase-6 <400> SEQUENCE: 49 ggatccatgg tagaaataga tgcagcctcc gtttac       #                   #       36 <210> SEQ ID NO 50 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for small s #ubunit of caspase-6 <400> SEQUENCE: 50 atcaattcaa cgtggtggtg gtggtggtgc          #                   #           30 <210> SEQ ID NO 51 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(5) <223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 51 Gln Ala Cys Xaa Gly  1               5 <210> SEQ ID NO 52 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 52 Asp Glu Val Asp  1 <210> SEQ ID NO 53 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 53 Ile Glu Thr Asp  1 <210> SEQ ID NO 54 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 54 Arg Thr Arg Thr Gly Ser  1               5 <210> SEQ ID NO 55 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 55 Leu Ser His Gly Cys Gln  1               5 <210> SEQ ID NO 56 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 56 Phe Ile Gln Ala Cys Gly Gly Glu Gln  1               5 <210> SEQ ID NO 57 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 57 Pro Glu Pro Asp Ala  1               5 <210> SEQ ID NO 58 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 58 Asp Gln Leu Asp Ala  1               5 <210> SEQ ID NO 59 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 59 Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gl #y Ser Trp Tyr Val  1               5   #                10   #                15 <210> SEQ ID NO 60 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 60 Arg Asp Arg Asn Gly Thr  1               5 <210> SEQ ID NO 61 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 61 Leu Ser His Gly Asp Lys  1               5 <210> SEQ ID NO 62 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 62 Phe Ile Gln Ala Cys Gln Gly Asp Asn  1               5 <210> SEQ ID NO 63 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 63 Val Glu Thr Asp Ser  1               5 <210> SEQ ID NO 64 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 64 Leu Glu Met Asp Leu  1               5 <210> SEQ ID NO 65 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 65 Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gl #y Thr Trp Tyr Ile  1               5   #                10   #                15 <210> SEQ ID NO 66 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 66 Lys Asp Arg Gln Gly Thr  1               5 <210> SEQ ID NO 67 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 67 Leu Thr His Gly Arg Phe  1               5 <210> SEQ ID NO 68 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 68 Phe Ile Gln Ala Cys Gln Gly Glu Glu  1               5 <210> SEQ ID NO 69 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 69 Ile Glu Ala Asp Ala  1               5 <210> SEQ ID NO 70 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 70 Gly Tyr Val Ser Phe Arg His Val Glu Glu Gl #y Ser Trp Tyr Ile  1               5   #                10   #                15 <210> SEQ ID NO 71 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 71 Gly Val Arg Asn Gly Thr  1               5 <210> SEQ ID NO 72 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 72 Leu Ser His Gly Glu Glu  1               5 <210> SEQ ID NO 73 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 73 Phe Ile Gln Ala Cys Arg Gly Thr Glu  1               5 <210> SEQ ID NO 74 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 74 Ile Gln Ala Asp Ser  1               5 <210> SEQ ID NO 75 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 75 Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gl #y Ser Trp Phe Val  1               5   #                10   #                15 <210> SEQ ID NO 76 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 76 Pro Glu Arg Arg Gly Thr  1               5 <210> SEQ ID NO 77 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 77 Leu Ser His Gly Glu Gly  1               5 <210> SEQ ID NO 78 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 78 Ile Ile Gln Ala Cys Arg Gly Asn Gln  1               5 <210> SEQ ID NO 79 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 79 Asp Val Val Asp Asn  1               5 <210> SEQ ID NO 80 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 80 Thr Glu Val Asp Ala  1               5 <210> SEQ ID NO 81 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 81 Gly Tyr Tyr Ser His Arg Glu Thr Val Asn Gl #y Ser Trp Tyr Ile  1               5   #                10   #                15 <210> SEQ ID NO 82 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 82 Thr Ser Arg Ser Gly Thr  1               5 <210> SEQ ID NO 83 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 83 Leu Ser His Gly Glu Glu  1               5 <210> SEQ ID NO 84 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 84 Ile Ile Gln Ala Cys Arg Gly Thr Glu  1               5 <210> SEQ ID NO 85 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 85 Ile Glu Thr Asp Ser  1               5 <210> SEQ ID NO 86 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 86 Gly Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gl #y Ser Trp Phe Ile  1               5   #                10   #                15 <210> SEQ ID NO 87 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 87 Pro Thr Arg Asn Gly Thr  1               5 <210> SEQ ID NO 88 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 88 Leu Ser His Gly Glu Glu  1               5 <210> SEQ ID NO 89 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 89 Phe Val Gln Ala Cys Arg Gly Glu Arg  1               5 <210> SEQ ID NO 90 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 90 Asp Ser Val Asp Gly  1               5 <210> SEQ ID NO 91 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 91 Gln Tyr Val Ser Trp Arg Asn Ser Ala Arg Gl #y Ser Trp Phe Ile  1               5   #                10   #                15 <210> SEQ ID NO 92 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 92 Pro Arg Arg Thr Gly Ala  1               5 <210> SEQ ID NO 93 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 93 Met Ser His Gly Ile Arg  1               5 <210> SEQ ID NO 94 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 94 Ile Ile Gln Ala Cys Arg Gly Asp Ser  1               5 <210> SEQ ID NO 95 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 95 Trp Phe Lys Asp Ser  1               5 <210> SEQ ID NO 96 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 96 Phe Glu Asp Asp Ala  1               5 <210> SEQ ID NO 97 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 97 Asp Asn Val Ser Trp Arg His Pro Thr Met Gl #y Ser Val Phe Ile  1               5   #                10   #                15 <210> SEQ ID NO 98 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 98 Pro Pro Arg Asn Gly Ala  1               5 <210> SEQ ID NO 99 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 99 Met Ser His Gly Ile Leu  1               5 <210> SEQ ID NO 100 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 100 Ile Val Gln Ala Cys Arg Gly Ala Asn  1               5 <210> SEQ ID NO 101 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 101 Trp Val Lys Asp Ser  1               5 <210> SEQ ID NO 102 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 102 Leu Glu Glu Asp Ala  1               5 <210> SEQ ID NO 103 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 103 His Asn Val Ser Trp Arg Asp Ser Thr Met Gl #y Ser Ile Phe Ile  1               5   #                10   #                15 <210> SEQ ID NO 104 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 104 Pro Ala Arg Asn Gly Ala  1               5 <210> SEQ ID NO 105 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 105 Met Ser His Gly Ile Leu  1               5 <210> SEQ ID NO 106 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 106 Ile Val Gln Ala Cys Arg Gly Glu Lys  1               5 <210> SEQ ID NO 107 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 107 Trp Val Arg Asp Ser  1               5 <210> SEQ ID NO 108 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 108 Leu Glu Ala Asp Ser  1               5 <210> SEQ ID NO 109 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 109 His Asn Val Ser Trp Arg Asp Arg Thr Arg Gl #y Ser Ile Phe Ile  1               5   #                10   #                15 <210> SEQ ID NO 110 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 110 Glu Phe Arg Ser Gly Gly  1               5 <210> SEQ ID NO 111 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 111 Leu Ser His Gly Val Glu  1               5 <210> SEQ ID NO 112 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 112 Phe Ile Gln Ala Cys Arg Gly Asp Glu  1               5 <210> SEQ ID NO 113 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 113 Asp Gln Gln Asp Gly  1               5 <210> SEQ ID NO 114 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 114 Glu Glu Ser Asp Ala  1               5 <210> SEQ ID NO 115 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 115 Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gl #y Ser Trp Tyr Ile  1               5   #                10   #                15 <210> SEQ ID NO 116 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Protease sensitive linker  #region between the       small and large subunits of a  #Rev-caspase <400> SEQUENCE: 116 Val Glu Ile Asp Ser  1               5 

I claim:
 1. A method of identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a sample containing an in vitro translated rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.
 2. A method of identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a cell transfected with a vector expressing rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.
 3. A method of identifying an inhibitor or enhancer of caspase-mediated apoptosis, comprising: (a) contacting a cell transfected with a vector expressing rev-caspase with a candidate inhibitor or candidate enhancer or with a reference compound; and (b) detecting cell viability, wherein viability of cells contacted with a candidate is increased in the presence of an inhibitor and is decreased in the presence of an enhancer compared to cells contacted with a reference compound.
 4. The method of any one of claims 1-3, wherein the rev-caspase is selected from the group consisting of rev-caspase-1, rev-caspase-2, rev-caspase-3, rev-caspase-4, rev-caspase-5, rev-caspase-6, rev-caspase-7, rev-caspase-8, rev-caspase-9, rev-caspase-10, rev-caspase-11, rev-caspase-12, rev-caspase-13, and rev-caspase-14.
 5. The method of either of claims 1 or 2, wherein detecting comprises gel electrophoresis.
 6. The method of claim 1, wherein the in vitro translated rev-caspase is labeled.
 7. The method of claim 6, wherein the label is a radioactive label, a peptide tag, an enzyme or biotin. 